Anti-annexin-V monoclonal antibodies, and preparation and use thereof

ABSTRACT

Using human annexin-V or human annexin-V plus dog annexin-V as antigen(s), hybridoma cell lines are prepared which are capable of producing anti-annexin-V monoclonal antibodies having a binding specificity to antigenic determinant site on annexin-V as antigenic protein and belonging to immunoglobulin G class. By the hybridoma cell lines are produced the anti-annexin-V monoclonal antibodies, with which a diagnostic agent is provided for diagnosis of myocardial infarction and angina pectoris. There is also provided diagnosis of myocardial infarction and angina pectoris using a first and a second monoclonal antibodies produced by the hybridoma cell lines to quantitate human annexin-V in a sample, in which an antigen-antibody reaction on annexin-V in the sample is caused with the first anti-annexin-V monoclonal antibody to form an annexin-V antigen/anti-annexin-V monoclonal antibody complex, the antigenic site of annexin-V of the formed annexin-V antigen/annexin-V monoclonal antibody complex is allowed to be bound with a labeled anti-annexin-V polyclonal or second monoclonal antibody so as to form a labeled form of said annexin-V antigen/anti-annexin-V monoclonal antibody complex bound with the polyclonal or the second monoclonal antibody, and the labeled form of the complex is quantitatively analyzed.

TECHNICAL FIELD

The present invention relates to anti-annexin-V monoclonal antibodieshaving binding specificities to antigenic determinant sites onannexin-V, a protein present in blood, plasma and/or serum of humans andmammalian animals, and, more particularly, to such anti-annexin-Vmonoclonal antibodies suitable for use in the immunologicaldetermination of the concentration of human annexin-V in the blood,plasma and/or serum, being present particulary in the human cardiacmuscles as a marker of myocardial infarction and angina pectoris.

The present invention further relates to hybridoma cell lines producingsaid anti-annexin-V monoclonal antibodies and, more particularly, tosuch hybridoma cell lines producing the monoclonal antibodies for use inthe detection and quantitative analysis of human cardiac-muscleannexin-V as a marker of myocardial infarction and angina pectoris.

The present invention also relates to a method for the detection ofannexin-V in a sample and, more particularly, to a method for thedetection or quantitative analysis of human cardiac-muscle annexin-V inthe blood and/or serum as a marker of myocardial infarction and anginapectoris. The present invention further relates to human cardiac muscleannexin-V in the plasma and/or serum, monoclonal antibodies for use inthe detection and quantitative analysis thereof and hybridoma producingsuch monoclonal antibodies.

In addition, the present invention relates to the development andutilization of anti-annexin-V antibodies for the measurement ofannexin-V of humans and mammals and the hybridoma cell lines producingsuch antibodies and, more particularly, to the development andutilization of the anti-human-annexin-V antibodies for use in quickdiagnosis of diseases causing sudden cellular necrosis such asmyocardial infarction and angina pectoris causing an ischemic disorder,through the concentration measurement of annexin-V, a protein in thecells or blood.

BACKGROUND ART

Annexin is a calcium-binding protein being present in tissues and cellsof humans and various animals, particularly in their cytoplasmicsolubles. This protein is composed of families defined by the amino acidsequences and, at present, annexin I through XII are known. The proteinbinds to phospholipids and actin depending upon the calciumconcentration, and is known to have anti-inflammatory and anticoagulantfunctions.

In patients suffering from tissue or cellular necrosis, due tomyocardial infarction, for example, various substances contained in thenecrosed cardiac-muscle cells leak into blood. The current diagnosis ofacute myocardial infarction is conducted by detecting such substances inthe blood and the substances are generally called myocardial-infarctionmarkers.

Such myocardial-infarction markers to be measured in biochemical testsfor use in the diagnosis of acute myocardial infarction include LD-1,AST, creatin kinase (CK), creatin kinase MB-fraction (CK-MB), myoglobin,lactic dehydrogenase (LDH), myosin light-chain I, and troponin-T (TnT).While the results or the analytical values of thesemyocardial-infarction markers may be employed independently or incombination in the diagnosis of myocardial-infarction, the prevailingmethod is to employ them in combination.

In the case of an ischemic disease such as angina pectoris, arrhythmiais observed arrhythmia within several hours after the onset of theattack, and may lead to arrhythmia death. Quick diagnosis and treatmentat the early stage are therefore needed in myocardial infarction andangina pectoris.

However, regarding the above-mentioned various types ofmyocardial-infarction markers (all are substances leaking from thecardiac muscles into the blood) to be examined in biochemical tests forthe diagnosis of acute myocardial-infarction, it has been reported thatsuch myocardial-infarction markers reach their respective peakconcentrations in the blood 7 to 78 hours after the attack of myocardialinfarction. In addition, the time in which the myocardial infarctionmarkers reach the maximal or peak concentration in the blood may varyconsiderably from one patient to another. Thus, these markers for thediagnosis of myocardial infarction are considered to be problematicsince they make it difficult to establish a quick diagnosis ofmyocardial infarction and hence they do not contribute to quickselection of therapeutic measures for the patients.

The employment of the results obtained by measuring two or more markersof the aforesaid myocardial-infarction markers for more reliablediagnosis of myocardial infarction is also problematic and far frommeeting the need for early-stage therapy of myocardial infarctionpatients because it takes a long time for the respectivemyocardial-infarction markers to reach the maximal (peak) concentrationsin the blood and such time varies depending upon the patient examined.

In the case of angina pectoris, marker substances to be examined inbiochemical tests for diagnozing the disease have not yet beenidentified. Thus, biochemical diagnosis of angina pectoris is generallyconsidered to be difficult and a need is felt for the establishment ofdiagnosis for angina pectoris through biochemical tests.

The present invention is directed to overcoming the problems in theconventional biochemical tests so as to enable quick diagnosis ofmyocardial infarction and angina pectoris.

DISCLOSURE OF THE INVENTION

It has been found that, among annexin-proteins present in human cardiacmuscles, human annexin-V, occurring in the solubles of thecardiac-muscle cells and having a molecular weight of 35,000, i.e.smaller than that of creatin kinase (81,000), generally leaks from thenecrosed cardiac muscles of myocardial-infarction or angina-pectorispatients in the early stage of the diseases. Thus the present applicanthas already disclosed a method for determining the concentration ofhuman annexin-V in human blood, plasma and/or serum and a method fordiagnosing myocardial infarction and/or angina pectoris, using dogcardiac-muscle annexin-V polyclonal antibodies which specificallycross-react with such human annexin-V (Japanese Laid-Open Pat.Application No.72147/1995).

Thus, human annexin-V is a novel marker of myocardial infarction whoseconcentration (in the blood, plasma and serum) reaches the peak withinone to several hours after the attack of myocardial infarction.

The present inventors therefore made extensive studies to provideantibodies capable of specifically recognizing human annexin-V andsuitable for use in a method for the assay of human annexin-V in asensitive, simple and quick manner and found that hybridoma cells,obtained from anti-human-annexin-V-producing lymphocytes and myelomacells using the conventional cell fusion technique, produce stableanti-human-annexin-V monoclonal antibodies meeting the above-mentionedobject for the completion of the present invention.

The present invention relates to an improvement on the invention of theaforesaid application and is directed to the provision of diagnosticagents and diagnostic methods, by which highly reliable diagnosis ofmyocardial infarction and/or angina pectoris can be made in a short timeafter the attack of myocardial infarction or angina pectoris, as well asthe provisions of detection substances for detecting and quantitativelyanalyzing the myocardial-infarction marker or angina pectoris marker andthe preparation and use thereof.

Thus, the present invention is directed to an anti-annexin-V monoclonalantibody characterized by having a binding specificity to antigenicdeterminant site on annexin-V as antigenic protein and belonging toimmunoglobulin G class, to a hybridoma cell line characterized by beingcapable of producing an anti-annexin-V monoclonal antibody having abinding specificity to antigenic determinant site on annexin-V asantigenic protein and belonging to immunoglobulin G class, to adiagnostic agent for myocardial infarction and angina pectorischaracterized by comprising an anti-annexin-V monoclonal antibody havinga binding specificity to antigenic determinant site on annexin-V asantigenic protein and belonging to immunoglobulin G class, and also to adiagnostic agent for myocardial infarction and angina pectorischaracterized by comprising a reagent containing a first anti-annexin-Vmonoclonal antibody having a binding specificity to antigenicdeterminant site on annexin-V as antigenic protein and belonging toimmunoglobulin G class and a reagent containing a second anti-annexin-Vmonoclonal or polyclonal antibody having a binding specificity toantigenic determinant site on annexin-V as antigenic protein andbelonging to immunoglobulin G class, with the second antibody beinglabeled.

In addition, the present invention is directed to a method fordiagnosing myocardial infarction and angina pectoris characterized bycomprising an antigen-antibody reaction of annexin-V in a sample with ananti-annexin-V monoclonal antibody to form an annexin-Vantigen/anti-annexin-V monoclonal antibody complex and assaying theformed annexin-V antigen/anti-annexin-V monoclonal antibody complex, toa method for diagnosing myocardial infarction and angina pectorischaracterized by comprising causing an antigen-antibody reaction ofannexin-V in a sample with an anti-annexin-V monoclonal antibody to forman annexin-V antigen/anti-annexin-V monoclonal antibody complex,allowing the antigenic site of annexin-V of the formed annexin-Vantigen/anti-annexin-V monoclonal antibody complex to bound with alabeled anti-annexin-V polyclonal or monoclonal antibody so as to form alabeled form of said annexin-V antigen/anti-annexin-V monoclonalantibody complex bound with the polyclonal or monoclonal antibody, andquantitatively analyzing the labeled form of the complex, to a methodfor analyzing human cardiac muscle or related annexin-V in a samplecharacterized by comprising causing an antigen-antibody reaction ofhuman annexin-V in the sample with an anti-annexin-V monoclonal antibodyto form an annexin-V antigen/anti-annexin-V monoclonal antibody complexand quantitatively analyzing the formed annexin-V antigen/anti-annexin-Vmonoclonal antibody complex, and also to a method for analyzingannexin-V in a sample characterized by comprising causing anantigen-antibody reaction of human annexin-V in the sample with a firstanti-annexin-V monoclonal antibody having a specificity to antigenicdeterminant site on annexin-V antigenic protein to form an annexin-Vantigen/anti-annexin-V monoclonal antibody complex, allowing theantigenic site of human annexin-V of the formed annexin-Vantigen/anti-annexin-V monoclonal antibody complex to be bound with ananti-annexin-V polyclonal or second monoclonal labeled antibody so as toform a labeled form of said annexin-V antigen/anti-annexin-V monoclonalantibody complex bound with the polyclonal or the second monoclonalantibody, and quantitatively analyzing the labeled form of the complex.

The present invention is further directed to a method for preparinganti-annexin-V monoclonal antibodies which comprises fusing lymphocytesinduced from mice immunized with annexin-V with myeloma cells to formhybridoma cells, and growing the formed hybridoma cells to produceanti-annexin-V monoclonal antibodies having binding specificities toantigenic determinant sites on annexin-V antigenic protein.

Human annexin-V to be used as the antigen in the present invention is aprotein which may have a molecular weight of 32 to 35 kilodalton and maybe one occurring in the human heart.

The human heart to be used in the present invention is one extractedfrom a human cadaver and maintaining its activities.

The antigen protein--human annexin-V protein occurring in humanheart--is found in the soluble fractions of the heart and purified byremoving the connective tissues and lipids from the cardiac tissue of ahuman cadaver. The hybridoma cell lines to be used in the presentinvention for producing anti-human annexin-V monoclonal antibodies areformed by means of the cell fusion of lymphocytic cells with myelomacells, in which the lymphocytic cells are spleen (a lymphoid organ)including spleen cells and white pulp of spleen or plasmablast cellsprepared from lymph nodes (e.g. lymphoid nodule) and being in theprocess of the differentiation to plasma cells, from a mammalian animalsuch as a mouse or the like which was immunized with human annexin-V asthe antigen extracted and purified from human cardiac tissue.

The myeloma cell strain to be used in the present invention is generallya hypoxanthine-guanine-phosphoribosyltransferase (HGPRT)-deficient oneor thymidine-kinase(TK)-deficient one. The HGPRT-deficient strainssuitable for use include 8-azaguanine(8-AG)-resistant strain and6-thioguanine(6-TG)resistant strain while the TK-deficient strainssuitable for use include bromodeoxyuridine(BUdR)-resistant strain.

The cell fusion is conducted using a cell fusion accelerator, in whichpolyethylene glycol (PEG) is primarily used for the cell fusion in thepresent invention.

The cell fusion accelerators usable in the present invention include, bysubstance name or trade name, PEG600, PEG400, PEG1000, PEG3000, PEG6000,GLUCAM™ E-10, GLUCAM™ E-20, GLUCAM™ P-10, polyethylene glycol methylester (PEG methyl ester) (molecular weight: 350, 2000 and 5000), PEG1000, polyglycol P15-200, pluronic F38 polyol Prilled, Decaglycerol,Camul 101 Butyrate, Tetronic 304 polyol, triglycerol,polyvinylpyrroridone (molecular weight: 1000) and glycerin.

The anti-human-annexin-V monoclonal antibodies in the present inventioncan be produced by fusing lymphocytic plasmablast cells extracted fromthe lymphoid organs of mice or other animals immunized against annexin-Vas the antigen with myeloma cells to form hybridomas and growing thehybridomas with, for example, a HAT culture medium, in which thehybridomas for the selective proliferation are, for example,anti-human-annexin-V-monoclonal-antibody-producing hybridoma cell linesHCA-627 and HDA-907 deposited on Aug. 16, 1994 and Nov. 7, 1995,respectively, at the National Institute of Bioscience andHuman-Technology, the Agency of Industrial Science and Technology ofJapan, an International Depositary Authority for the deposit ofmicroorganism, under the numbers FERM BP-5284 and FERM BP5286,respectively.

The selection of the hybridoma cell lines can be made through aHAT-selection, an ouabain-selection or the like.

The anti-human-annexin-V monoclonal antibodies in the present inventioncan be obtained by culturing the above-mentioned hybridoma clones by aconventional culture process such as the high density culture process,the spinner-flask culture process or the like and purifying theantibodies from the culture supernatant by means of affinitychromatography using a carrier bound with protein-A or a carrier boundwith anti-mouse-immunoglobulin.

In the present invention, the anti-human-annexin-V monoclonal antibodiescan be produced by injecting the hybridoma cell lines obtained throughthe above-mentioned culture into the abdominal cavities ofprestene-pretreated immunocompromised mice, salting out, with ammoniumsulfate or sodium sulfate, the ascitic fluid induced by the hybridomacell lines, and purifying the fluid by means of ion-exchangechromatography using DEAE cellulose to obtain the immunoglobulin (IgG)fractions.

A hybridoma cell line as termed with respect to the present inventionmeans not only a hybridoma cell just obtained by cell-fusion but alsohybridoma cells at any passages subsequent to the primary culture.

The screening for the resultant hybridoma cell lines can be conducted bydetecting the anti-human-annexin-V monoclonal antibodies secreted by thehybridomas in the culture by such methods as immunoassay,radioimmunoassay and ELISA.

In the present invention, the process of screening for the hybridomacell lines has been found to be able to obtain variousanti-human-annexin-V monoclonal antibodies having different specificreactivities with human-annexin-V antigen.

By a method similar to that for producing anti-human-annexin-Vmonoclonal antibodies there can be obtained anti-annexin-V monoclonalantibodies having specific reactivities with the antigenic determinantsites of the annexin-V antigens of dogs and other mammalian animals, byimmunizing the mammalian animals with the annexin-V antigens derivedfrom dogs or the like, cell-fusing the lymphocytic cells with myelomacells to produce the hybridoma cell lines.

The anti-annexin-V monoclonal antibodies in the present invention aredefined to include such anti-annexin-V monoclonal antibodies frommammalian animals as well as anti-human-annexin-V monoclonal antibodies.

Thus, cloning is carried out for the hybridoma cell lines which havebeen screened for the ability to secrete anti-human-annexin-V monoclonalantibodies.

The cloning of the hybridoma cell lines in the present invention can bemade by such techniques as limiting dilution, soft agar method, fibringel method, cell sorter method and the like.

The hybridoma cell lines with an ability to produce anti-human-annexin-Vmonoclonal antibodies thus obtained are subjected to a large-scalefermentation to produce anti-human-annexin-V monoclonal antibodies.

The anti-human-annexin-V monoclonal antibodies of the present inventionhave specific reactivities with the antigenic determinant sites of humanannexin-V, a protein antigen. They are antibodies featuring anantigen-antibody reaction with the antigenic protein (human annexin-V)and will thus bind to human annexin-V present in human blood, plasmaand/or serum, thereby enabling highly specific and sensitive detectionand quantitative analysis of human annexin-V in human blood, plasmaand/or serum through such techniques as immunoassay, radioimmunoassay,ELISA and the like.

The anti-human-annexin-V monoclonal antibodies of the present inventionexhibit specific reactivities with the antigenic determinant sites ofhuman annexin-V, a protein occurring in the human heart, therebyenabling highly specific and sensitive detection and/or quantificationof human annexin-V in the blood, plasma and/or serum, through suchtechniques as immunoassay, radioimmunoassay, ELISA and the like, as theybind to the antigenic protein, human annexin-V, present in the humanblood, plasma and/or serum.

While human annexin-V concentration in the blood of a normal adult isfound, for example, to be 5.6 ng/ml at the highest, it has been found bythe application of the present invention that the human annexin-Vconcentration with a patient suffering myocardial infarction may be, forexample, as high as 90 ng/ml even in the stage when the CK valueindicated is normal and also that the human annexin-V concentration of apatient suffering angina pectoris may be as high as 29 ng/ml even in thestage when the CK value indicated is normal. The present inventiontherefore enables a diagnosis of the diseases at earlier stages thanpossible with the conventional diagnosis based on the CK value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a calibration curve for use in ELISA system for aworking example, in which the anti-human-annexin-V monoclonal antibodiesof the present invention are employed to determine the concentration of8M-urea-treated human annexin-V. The ELISA system is made up of acombination of a HRPO-labeled anti-human-annexin-V monoclonal antibodyproduced by HCA-155H, an anti-human-annexin-V monoclonalantibody-producing hybridoma cell line clone, and having a specificactivity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-290, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 2 illustrates a calibration curve for use in ELISA system foranother working example, in which the anti-human-annexin-V monoclonalantibodies of the present invention are employed to determine theconcentration of 8M-urea-treated human annexin-V. The ELISA system ismade up of a combination of a HRPO-labeled anti-human-annexin-Vmonoclonal antibody produced by HCA-155H, an anti-human-annexin-Vmonoclonal antibody-producing hybridoma cell line clone, and having aspecific activity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-57HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 3 illustrates a calibration curve for use in ELISA system for afurther working example different from the examples as shown by FIGS. 1and 2, in which the anti-human-annexin-V monoclonal antibodies of thepresent invention are employed to determine the concentration of8M-urea-treated human annexin-V. The ELISA system is made up of acombination of a HRPO-labeled anti-human-annexin-V monoclonal antibodyproduced by HCA-155H, an anti-human-annexin-V monoclonalantibody-producing hybridoma cell line clone, and having a specificactivity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-293HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length thearrows extending upward or downward from each □ mark represents theaverage ±2 SD respectively.

FIG. 4 illustrates a calibration curve for use in ELISA system for astill further working example different from the examples as shown byFIGS. 1 through 3, in which the anti-human-annexin-V monoclonalantibodies of the present invention are employed to determine theconcentration of 8M-urea-treated human annexin-V. The ELISA system ismade up of a combination of a HRPO-labeled anti-human-annexin-Vmonoclonal antibody produced by HCA-155H, an anti-human-annexin-Vmonoclonal antibody-producing hybridoma cell line clone, and having aspecific activity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-350HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 5 illustrates a calibration curve for use in ELISA system for aworking example different from the examples as shown by FIGS. 1 through4, in which the anti-human-annexin-V monoclonal antibodies of thepresent invention are employed to determine the concentration of.8M-urea-treated human annexin-V. The ELISA system is made up of acombination of a HRPO-labeled anti-human-annexin-V monoclonal antibodyproduced by HCA-660HD, an anti-human-annexin-V monoclonalantibody-producing hybridoma cell line clone, and having a specificactivity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-290HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 6 illustrates a calibration curve for use in ELISA system for afurther working example different from the examples as shown by FIGS. 1through 5, in which the anti-human-annexin-V monoclonal antibodies ofthe present invention are employed to determine the concentration of8M-urea-treated human annexin-V. The ELISA system is made up of acombination of a HRPO-labeled anti-human-annexin-V monoclonal antibodyproduced by HCA-660HD, an anti-human-annexin-V monoclonalantibody-producing hybridoma cell line clone, and having a specificactivity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-57HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 7 illustrates a calibration curve for use in ELISA system for astill further working example different from the examples as shown byFIGS. 1 through 6, in which the anti-human-annexin-V monoclonalantibodies of the present invention are employed to determine theconcentration of 8M-urea-treated human annexin-V. The ELISA system ismade up of a combination of a HRPO-labeled anti-human-annexin-Vmonoclonal antibody produced by HCA-660HD, an anti-human-annexin-Vmonoclonal antibody-producing hybridoma cell line clone, and having aspecific activity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-293HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 8 illustrates a calibration curve for use in ELISA system foranother working example different from the examples as shown by FIGS. 1through 7, in which the anti-human-annexin-V monoclonal antibodies ofthe present invention are employed to determine the concentration of8M-urea-treated human annexin-V. The ELISA system is made up of acombination of a HRPO-labeled anti-human-annexin-V monoclonal antibodyproduced by HCA-660HD, an anti-human-annexin-V monoclonalantibody-producing hybridoma cell line clone, and having a specificactivity with human annexin-V, and another anti-human-annexin-Vmonoclonal antibody as the solid phase, produced by HCA-350HDR, anotheranti-human-annexin-V monoclonal antibody-producing hybridoma cell lineclone, and having a specific activity with human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which eachmark represents the average difference absorbance with the arrowsextending upward or downward from each □ mark represents the average±2SD respectively.

FIG. 9 illustrates a calibration curve for use in ELISA system for aworking example, in which the anti-human-annexin-V monoclonal antibodiesof the present invention are employed to determine the concentration ofnative human annexin-V. The ELISA system is made up of a combination ofa HRPO-labeled anti-dog-32KP polyclonal antibody, and ananti-human-annexin-V monoclonal antibody as the solid phase, produced byHCA-627, an anti-human-annexin-V monoclonal antibody-producing hybridomacell line clone, deposited under the number FERM BP-5284 at theInternational Depositary Authority for the deposit of microorganisms andhaving a specific activity with native human annexin-V.

In the Figure, the ordinate indicates difference absorbance obtained bysubtracting an absorbance measurement at the secondary wavelength 690 nmfrom an absorbance measurement at the primary wavelength 492 nm and theabscissa indicates the concentration of human annexin-V, in which each □mark represents the average difference absorbance and the length of thearrows extending upward or downward from each □ mark represents theaverage ±2SD respectively.

FIG. 10 is a microscopic photograph, under 200 diameter magnification,of human cardiac muscle tissue subjected to a specific staining byindirect enzyme antibody technique in which anti-human-annexin-Vmonoclonal antibody HCA-627 was employed as the primary antibody andHRPO-labeled-anti-mouse-immunoglobulin antibody was employed as thesecondary antibody. The figure demonstrates that human annexin-V wasstained in the form of uneven aggregates in the cytoplasm within thehuman heart of a patient with sudden death.

FIG. 11 is a microscopic photograph, under a 200 diameter magnification,of human cardiac muscle tissue for comparison with the example as shownby FIG. 10. The figure demonstrates that without the use of theanti-human-annexin-V monoclonal antibody, it is impossible to stainhuman annexin-V and therefore to locate the distribution thereof.

FIG. 12 illustrates a calibration curve for use in ELISA system for aworking example, in which the anti-human-annexin-V monoclonal antibodiesof the present invention are employed to determine the concentration ofnative human annexin-V. The ELISA system is made up of a combination ofa HRPO-labeled anti-annexin-V monoclonal antibody, produced by HDA-907,an anti-annexin-V monoclonal antibody-producing hybridoma cell lineclone, deposited under the number FERM BP-5286 at the InternationalDepositary Authority for the deposit of microorganisms and having aspecific activity with native human annexin-V, and another anti-humanannexin-V monoclonal antibody as the solid-phase, produced by HCA-627,an anti-human-annexin-V monoclonal antibody-producing hybridoma cellline clone and having a specific activity with native human annexin-V.

In the Figure, the ordinate indicates absorbance obtained by subtractingan absorbance measurement at the secondary wavelength 690 nm from anabsorbance measurement at the primary wavelength 492 nm and the abscissaindicates the concentration of human annexin-V, in which each ◯ markrepresents the average difference absorbance measured four times and thelength of the arrows extending upward or downward from each ◯ markrepresents the average ±2SD respectively.

FIG. 13 shows the results of dilution tests performed on plasma samplesby an ELISA system for assay of native human annexin-V concentrationusing the anti-annexin-V monoclonal antibodies of the present invention.The ELISA system was constructed using a combination of HRPO-labeledanti-human-annexin-V monoclonal antibody produced by anti-annexin-Vmonoclonal antibody-producing hybridoma cell line clone HDA-907(deposited under the number FERM BP-5286 at the International DepositaryAuthority for deposit of microorganism) and having a specific reactivitywith annexin-V and anti-human-annexin-V monoclonal antibody as solidphase produced by anti-annexin-V monoclonal antibody-producing hybridomacell line clone HCA-627 (deposited under the number FERM BP-5284 at theInternational Depositary Authority for deposit of microorganism) andhaving a specific reactivity with native human annexin-V.

In the Figure, the ordinate shows human annexin-V concentration measuredon the original solution and the series of diluted solution of eachsample and the abscissa represents 1/dilution rate for each sample.

THE BEST MODE FOR CARRYING OUT THE INVENTION

While the present invention will be further illustrated by the examplesgiven below, the invention is not to be limited by the followingexamples and description.

In the following examples the symbol M represents molarity and itindicates, for a solution of mixture, the mol per liter of the solution.

EXAMPLE 1 Hybridoma Preparation

(1) Purification of Human Annexin-V

The heart was excised from a human adult cadaver. After clearing theblood, the right ventricle was excised from the heart, followed by theremoval of the connective tissue and the lipids therefrom. Theseoperations were carried out in ice or at a 4° C. temperature.

The heart was added with a buffer solution of the following compositionin a ratio of ten times as much of the heart weight: 250 mM, sucrose 0.5mM ethylene glycol bis(2-aminoethyl ether) tetraacetate (EGTA), 1 mMphenylmethane sulfonyl fluoride (PMSF) and 10 mM tris(tris(hydroxymethyl) aminomethane)HCl, pH7.4.

The heart was then homogenized by a homogenizer and the homogenate wascentrifuged at 3000 xg for 15 minutes. The separated supernatant isadded with 1M CaCl₂ solution to give a final concentration of 2 mM formixing, followed by centrifugation at 28,000 xg for an hour. To theresidue was added 2 ml of 10 mM EGTA for suspending the residue. Theresidue suspension was centrifuged at 28,000 xg for 1 hour.

The supernatant was subjected to gel filtration with Sephacryl S-300(trade name) column available from Pharmacia Co. for elution with pH 7.4buffer solution (B) containing 0.1M NaCl and 30 mM Tris-HCl. Thefraction containing protein having a molecular weight of 35 KDa wasrecovered and passed through an ion-exchange column (Biogelagarose:trade name) with an eluent of 10 mM Tris HCl solution (pH 7.4)containing NaCl in the concentration range of 0 to 0.3M for purificationby means of NaCl-concentration-gradient elution.

The human annexin-V thus purified was divided into two portions, one ofwhich was freeze-dried and then dissolved by 0.1M sodium phosphatebuffer (pH 7.6) containing 8M urea. The other part was freeze-dried anddissolved by 0.1M sodium phosphate buffer (pH 7.6). The two parts wereboth preserved at 4° C. These parts of purified human annexin-V weremeasured for purity with sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) to quantify the protein concentration.

Identification was also made of the purified annexin-V protein: Peptidefractions were prepared by adding lysylendopeptidase and keeping themixture at 37° C. for 15 hours so as to allow the human annexin-Vprotein to react with lysylendopeptidase. The peptides thus obtainedwere analyzed for amino acid sequence on a Shimadzu PPSQ-10 proteinsequencer to determine the amino acid sequences of the two peptidefractions, based on Edman method (cf. Edman P. "A method for thedetermination of the amino acid sequence in peptide", Arch. Biochem,Biophys., 1949, Vol.22, page 475).

As the N-terminal amino acid sequences of the two peptides from thepurified protein are Glu-Tyr-Gly-Ser-Ser-Leu-Glu (SEQ ID NO:1) for thefirst and Gly-Thr-Asp-Glu-Glu-Lys-Phe-Ile-Thr-Ile-Phe-Gly-Thr (SEQ IDNO:2) for the second, the protein was identified as annexin-V.

(2) Mice

BALB/c inbred-strain female mice 5 to 8 weeks of age were maintained ona diet of standard pellets feeding water ad libitum in ananimal-breeding chamber kept at the temperature of 23°+1° C. and thehumidity of 70%.

(3) Immunization

The human annexin-V 100 micrograms/0.5 ml as prepared in (1) was mixedwith an equal amount of Freund's complete adjuvant to be emulsified. Thehuman annexin-V in emulsion was administered as the antigen into theabdominal cavities of four female mice 5 weeks of age in a dose of 15-40micrograms of the purified annexin-V per mouse. The administration wasmade every two weeks for two months for the immunization of the mice.The mice were determined for the antibody titer to select mice having ahigh titer, which were administered, three weeks after, with 50micrograms of the purified human annexin-V as a booster intravenouslythrough the mice tails for final immunization.

(4) Cell Fusion

Three days after final immunization, the spleens were excised fromBALB/c mice and the spleen cells were suspended in EMEM culture mediumto prepare a suspension of spleen cells. The spleen cells were thenwashed four times with EMEM medium (available from Nissui Co.) anddetermined for the number of cells. The result was that 7×10⁸ livingspleen cells were obtained.

Cell fusion was conducted using P3-X63-Ag8 653 culture cells,hereinafter referred to as X63 cells, as parent cell strain, which are2-amino-6-oxy-8-azapuraine (8-azaguanine)-resistant and derived fromBALB/c mouse myeloma. X63 cells in the logarithmic growth phase wereemployed to subculture in RPMI-1640 medium (at a concentration of 20micrograms/ml and with 8-azaguanine) (available from GIBCO Co.),containing 10% of immobilized fetal calf serum (hereinafter referred toas FCS) available from Intergen Co. Further cultivation was conducted,from three days prior to the cell fusion, in RPMI-1640 medium containing10% of FCS but not containing 8-azaguanine, in which the cells in thelogarithmic growth phase were also employed. Following washing withRPMI-1640 medium three times, X63 cells were determined for the numberof cells, with the result that the number of living X63 cells was 7×10⁷.

Polyethylene glycol-4000 available from Sigma Co. was dissolved inRPMI-1640 medium to give a concentration of 50% (w/v) for use in thecell fusion.

The spleen cells and X63 cells were mixed in a ratio of the spleencells: X63 cells=10:1. The mixture was centrifuged at 1500 rpm for fiveminutes, the supernatant was removed and the cells were welldisintegrated to be used in the cell fusion. The cell fusion was carriedout, using polyethylene glycol prepared in the aforesaid manner and keptat 37° C., in accordance with the method described in "Kohller andMilstein: Nature, Vol.256, pages 495-497 (1975)" and "European Journalof Immunology, Vol.6, pages 511-519 (1976)".

The cell lines following the cell fusion for the hybridoma formationwere suspended in HAT selection medium containing RPMI-1640 added withFCS of a 10% concentration, 1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ M aminopterineand 1.6×10⁻⁵ M thymidine, to give a spleen cell concentration of 2.0×10⁶per ml. Each 50 microliters aliquot of the cell suspension wasdistributed in 96 wells, and incubated an incubator kept at 37° C.temperature and 95% humidity under a 8% CO₂ atmosphere. One drop of theHAT medium was added to each well on the first day and the second dayfrom the start of the incubation, followed by the addition of two dropsof the same medium on the seventh and ninth day from the start forfurther incubation. Then incubation was carried out in a HAT-freeculture medium. Approx. ten days to two weeks after the start, screeningwas made for hybridoma cell clones producing anti-human-annexin-Vmonoclonal antibodies capable of specifically reacting with humanannexin-V treated with urea of 8M concentration (8M-urea-treated humanannexin-V) and/or with human annexin-V not treated with 8M urea (nativehuman annexin-V). The screening was done by ELISA using a humanannexin-V absorption test and microplates in which human annexin-V asthe antigenic protein is adsorved onto a solid phase.

All the clones of established hybridoma cell lines producinganti-human-annexin-V monoclonal antibodies as well as anti-dog-32KPpolyclonal antibody are sensitized for solid phase. In addition, theanti-human-annexin-V monoclonal antibodies produced from such clones aswell as anti-dog-32KP polyclonal antibody are labeled with biotin. Thus,a variety of ELISA methods were employed: the solid-phase-coupled clonesand anti-dog-32KP polyclonal antibody are used in combination with thebiotin-labeled anti-human-annexin-V monoclonal antibodies and alsoanti-dog-32KP polyclonal antibody, in order to detect clone(s) producinganti-human-annexin-V monoclonal antibodies which will specifically reactwith 8M-urea-treated human annexin-V, native human annexin-V and nativehuman annexin-V in the human blood, serum or plasma not treated with 8Murea.

(5) Screening

As cell clones appeared ten days after the start of incubation, anabsorption test with human annexin-V as the antigen was carried out byELISA for the supernatants of hybridoma cell lines.

It was found by the present inventors that the antigenic activity ofhuman annexin-V treated with 8M urea solution is significantly higherthan that of native human annexin-V, with anti-32KP polyclonalantibodies described later. Thus, the human annexin-V absorption testwas carried out using the two types of antigens: human annexin-V treatedwith 8M urea (hereinafter referred to as (8M-urea-treated humanannexin-V) and human annexin-V with no such urea-treatment (hereinafterreferred to as native human annexin-V).

Thus, a first set is constituted of 50 microliters per well of8M-urea-treated human annexin-V solution (the 8M-urea-treated humanannexin-V concentration being 200 ng/ml) and 50 microliters per well ofthe culture supernatant of hybridoma cell line distributed eachU-bottomed well of a microtiterplate, while a second set is constitutedof 50 microliters per well of native human annexin-V (the native humanannexin-V concentration being 200 ng/ml) and 50 microliters per well ofthe supernatant of hybridoma cell line distributed in each U-bottomedwell of another microtiterplate. Each well was added with 50 microlitersof 20% suspension of Sepharose 4B combined withanti-mouse-immunoglobulin antibody and allowed to stand for 10 minutesfollowing stirring for one hour. With each set, when it was observedthat the anti-mouse-immunoglobulin antibody-combined Sepharose 4Bcompletely sedimented on the well bottom, the respective supernatants 25microliters were employed to measure remaining human annexin-Vconcentration therein by means of the human annexin-V ELISA method.

In this measurement, if any anti-human-annexin-V monoclonal antibodiesagainst human annexin-V was present in the culture supernatant ofhybridoma cell line, reactions will occur between human annexin-V andthe anti-human-annexin-V monoclonal antibodies, followed by a reactionwith anti-mouse-immunoglobulin monoclonal antibody-combined Sepharose4B, resulting in the formation of antigen-antibody complexes which areto sediment. This will lead to a decrease in the human annexin-V contentremaining in the supernatant and it is therefore possible to identifythe presence of any anti-human-annexin-V monoclonal antibodies throughthe measurement of the human annexin-V concentration in the supernatant.

After the screening was completed in the above-mentioned manner foranti-human-annexin-V monoclonal antibodies capable of reacting withhuman annexin-V as the antigenic protein, a test was conducted to studythe difference in epitopes with which the anti-human-annexin-Vmonoclonal antibodies will react.

Thus, 8M-urea-treated human annexin-V and native annexin-V as theantigenic solutions, each having been prepared to have a concentrationof 1 microgram/ml, were absorbed onto a microtiterplate in an amount of50 microliters per well, followed by washing three times with phosphatebuffer solution (hereinafter referred to as PBS) containing 0.05%Tween-20, a Tween surfactant, and blocking with PBS containing 1% BSA toprepare plates with solid phase-coupled human annexin-V antigens.

A solid phase-coupled human annexin-V antigen in the plates thusobtained was allowed to react, for one hour at room temperature, with aculture supernatant of the hybridoma cell lines producing monoclonalantibodies capable of reacting with human annexin-V screened by thehuman annexin-V absorption test. The resulting antigen-antibodycomplexes were subjected to three times of washing with the washingsolution and allowed to react, for thirty minutes at room temperature,with anti-mouse-immunoglobulin antibodies (from goats) labeled withhorseradish peroxidase (hereinafter referred to as HRPO). Following thereaction, the resultant antigen-antibody complexes were subjected towashing four times with the washing solution and allowed to react, atroom temperature for five minutes, with OPD substrate solutioncontaining 0.1M phosphate-citrate buffer solution, 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂, and then the reaction was terminatedwith 2N H₂ SO₄ and absorbance measurement was performed by the dualwavelength method at 492 nm as the primary wavelength and at 690 nm asthe secondary wavelength on an ELISA plate reader.

At the primary wavelength 492 nm absorbance is measured with thesubstrate undergone the reaction due to the bound labeled-antibodies,while at the secondary wavelength the absorbance reflects cracks orstains of the microtiterplates. Accordingly, the subtraction of theabsorbance at the secondary wavelength from that at the primarywavelength makes it possible to determine the absorbance reflecting theamount of the enzyme on the labeled antibodies which have bound to thesolid phase-coupled antigen in proportion to the amount of the antigen.

(6) Studies on Reactivities with Solid Phases Sensitized with Annexin-VAntigens from Various Organs and Animals

Studies were made of the anti-human-annexin-V monoclonal antibodies thusobtained for the species-dependent specificities with annexin-V derivedfrom various human organs and animals, using the solid phases sensitizedin the above-mentioned manner for human annexin-V.

With each annexin purified from the various human organs and animals, asolution was prepared to give a concentration of 1 microgram/ml with0.1M phosphate buffer at pH 7.5. 50 microliters of each antigen solutionwas distributed in a 96-well microtiterplate for the adsorptionovernight at 4° C., followed by washing twice with the washing solutionand blocking with the blocking solution. The solid phases sensitizedwith the various annexin-V antigens were allowed to react with theanti-human-annexin-V monoclonal antibodies obtained by the exampleherein. The reaction products were assayed by the ELISA method in thesame manner for the screening, for determining the reactivities of theanti-human-annexin-V monoclonal antibodies as obtained by the presentinvention with the respective annexin-V antigens.

(7) Studies on Reaction Specificities by Western Blotting

Homogenized specimens of the human cardiac muscle, the human liver, thehuman kidney, the human lungs, the beagle's cardiac muscle and the rat'scardiac muscle were subjected to sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) using 10 to 20% polyacrylamide gel tofractionate the protein fractions. The protein fractions are thentransferred into nitrocellulose membranes, followed by a blocking withPBS containing 3% skim milk and 1% BSA. Then the protein fractions fixedonto the membranes were allowed to react with the respective monoclonalantibodies to examine reactive protein fractions derived from thevarious organs and animals.

(8) Establishment of Hybridoma Cell Lines Producing MonoclonalAntibodies

Based on the above-mentioned studies, eighty-four strains were screenedwhich are hybridoma cell line clones capable of reacting with8M-urea-treated human annexin-V, through the absorption test using humancardiac muscle annexin-V. From such strains twenty-one clone strainswere strictly selected depending upon the difference in reactivitieswith type of annexin-V derived from the various organs and animals.Thus, the twenty-one strains were classified into the following fivegroups depending upon the reactivities:

(i) Clones which exhibit a high reactivity in the 8M-urea-treated humancardiac muscle annexin-V absorption test, but do not exhibitreactivities with any of the antigen-sensitized solid phases: fivestrains (clone HCA-212, clone HCA-290, clone HCA-656, clone HCA-713 andclone HCA-805).

(ii) Clones which exhibit a high reactivity in the 8M-urea-treated humancardiac muscle annexin-V absorption test and also exhibit a highreactivity with the human-annexin-V-sensitized solid phase, but exhibitalmost no or a low reactivity with the beagle dog- andrat-derived-annexin-V-sensitized solid phases: eight strains (cloneHCA-69H, clone HCA-155H, clone HCA-231H, clone HCA-671H, clone HCA-770H,clone HCA-784H, clone HCA-803H and clone HCA-838H).

(iii) Clones which exhibit high reactivities in the 8M-urea-treatedhuman cardiac muscle annexin-V absorption test, as well as with all ofthe solid phases sensitized with annexin-V antigens derived from thevarious human organs, the beagle's and the rat's cardiac muscles: fourstrains (clone HCA-57HDR, clone HCA-293HDR, clone HCA-350HDR and cloneHCA-805HDR).

(iv) Clones which exhibit high reactivities in the 8M-urea-treated humancardiac muscle annexin-V absorption test and also with the solid phasessensitized with annexin-V antigens from the human cardiac muscle, thehuman kidney or the human liver, but exhibit only low reactivities withthe solid phases sensitized with annexin-V antigens from the beagle'sand the rat's cardiac muscles: three strains (clone HCA-507HD, cloneHCA-660HD and clone HCA-646HD).

(v) A clone which exhibit high reactivities in the 8M-urea-treated humancardiac muscle annexin-V absorption test as well as in the native humancardiac muscle annexin-V absorption test: one strain (clone HCA-627).

Thus, there were obtained monoclonal antibodies having differentreactivities as classified into the five groups mentioned in the above.

The reaction specificities of these monoclonal antibodies are shown inTable 1 below.

                                      TABLE 1                                     __________________________________________________________________________    Reaction Specificities of Anti-human-cardiac-muscle-annexin-V Monoclonal      Antibodies                                                                    (Absorbance = A4902 nm 690 nm)                                                __________________________________________________________________________    Cell fusion No.                                                               1              Absorption Rate (%)                                                                    Absorption Rate (%)                                   Anti-human-                                                                          Mouse   in 8M-urea-treated                                                                     in Native Human                                                                        Absorption Rate (%)                          cardiac-muscle                                                                       Immunogulobulin                                                                       Human-cardiac-                                                                         cardiac-muscle                                                                         in 8M-urea-treated                           annexin-V                                                                            lsotype muscle Annexin-V                                                                       Annexin-V Antigen                                                                      Dog Annexin-V Antigen                        Mab clone No.                                                                        H-chain                                                                           L-chain                                                                           Absorption Test                                                                        Absorption Test                                                                        Absorption Test                              __________________________________________________________________________    HCA-212                                                                              IgG1                                                                              k   85       40       64                                           HCA-290                                                                              IgG1                                                                              k   82       10       86                                           HCA-656                                                                              IgG1                                                                              k   67       1        57                                           HCA-713                                                                              IgG2a                                                                             k   47       4        29                                           HCA-805                                                                              IgG1                                                                              k   83       38       55                                           HCA-69H                                                                              IgG2b                                                                             k   87       3        4                                            HCA-155H                                                                             IgG2a                                                                             k   89       18       31                                           HCA-231H                                                                             IgG2a                                                                             k   90       6        24                                           HCA-671H                                                                             IgG2a                                                                             k   91       9        29                                           HCA-770H                                                                             IgG2b                                                                             k   88       7        26                                           HCA-784H                                                                             IgG2a                                                                             k   88       3        26                                           HCA-803H                                                                             IgG2a                                                                             k   88       5        20                                           HCA-838H                                                                             IgG2a                                                                             k   88       6        10                                           HCA-507HD                                                                            IgG2a                                                                             k   90       7        36                                           HCA-660HD                                                                            IgG1                                                                              k   90       9        42                                           HCA-646HD                                                                            IgG2b                                                                             k   91       5        35                                           HCA-57HDR                                                                            IgG2a                                                                             k   62       7        46                                           HCA-293HDR                                                                           IgG2a                                                                             k   66       5        55                                           HCA-350DHR                                                                           IgG2a                                                                             k   65       3        52                                           HCA-805HDR                                                                           IgG1                                                                              k   57       4        45                                           HCA-627                                                                              IgG1                                                                              k   99       99       87                                           reagent blank  0        0        0                                            __________________________________________________________________________    Reactivity with Solid-phase of                                                              Reactivity with Solid-phase of                                                               Reactivity with Solid-phase of                   Human Cardiac Muscle-derived                                                                Human Kidney-derived                                                                         Human Liver-derived                              Annexin-V Antigen                                                                           Annexin-V Antigen                                                                            Annexin-V Antigen                                (1 μg/ml Antigen sensitized                                                              (1 μg/ml Antigen sensitized                                                               (1 μg/ml Antigen sensitized                   at 50 μl/well)                                                                           at 50 μl/well)                                                                            at 50 μl/well)                                Absorbance in Absorbance in                                                   8M-urea-treat-                                                                       Absorbance in                                                                        8M-urea-treated                                                                       Absorbance in                                                                        Absorbance in                                                                         Absorbance in                            ed Antigen                                                                           Native Antigen                                                                       Antigen Native Antigen                                                                       8M-urea-treated                                                                       Native Antigen                           Sensitization                                                                        Sensitization                                                                        Sensitization                                                                         Sensitization                                                                        Antigen Sensitization                            __________________________________________________________________________    0.009  0.079  0.009   0.026  0.033   0.011                                    0.145  0.080  0.018   0.026  0.045   0.008                                    0.119  0.131  0.012   0.040  0.109   0.010                                    0.023  0.080  0.015   0.006  0.007   0.005                                    0.014  0.096  0.010   0.021  0.029   0.006                                    9.460  13.240 4.610   4.420  1.191   2.712                                    11.330 14.650 6.670   7.520  2.074   4.690                                    9.230  14.260 5.110   5.060  1.529   3.160                                    8.900  15.680 6.950   7.850  2.421   5.620                                    11.260 12.030 3.680   3.560  1.077   2.268                                    9.860  14.080 5.580   5.860  1.854   4.210                                    12.730 14.440 5.890   6.580  2.269   5.270                                    10.450 13.860 4.630   5.000  1.508   3.190                                    10.110 15.940 7.420   8.840  3.090   6.710                                    13.540 15.170 7.300   8.930  3.300   6.340                                    8.360  13.240 5.530   6.390  1.986   4.190                                    9.910  8.140  2.459   1.465  0.531   0.464                                    12.330 12.590 3.740   3.290  0.923   1.156                                    12.510 9.190  3.180   2.305  0.729   0.853                                    2.499  0.766  0.096   0.063  0.048   0.051                                    0.042  0.224  0.036   0.066  0.193   0.015                                    0.041  0.045  0.014   0.015  0.009   0.010                                    __________________________________________________________________________    Reactivity with Solid-phase of                                                                      Reactivity with Solid-phase of                          Rat Cardiac Muscle-derived                                                                          Beagle Cardiac Muscle-derived                           Annexin-V Antigen     Annexin-V Antigen                                       (1 μg/ml Antigen Sensitized at 50 μl/well)                                                    (1 μg/ml Antigen Sensitized at 50 μl/well)        Absorbance in 8M-urea-                                                                              Absorbance in 8M-urea-                                  treated Antigen                                                                          Absorbance in Native                                                                     treated Antigen                                                                          Absorbance in Native                         Sensitization                                                                            Antigen Sensitization                                                                    Sensitization                                                                            Antigen Sensitization                        __________________________________________________________________________    0.008      0.006      0.016      0.020                                        0.043      0.080      0.126      0.670                                        0.367      0.371      0.318      1.664                                        0.031      0.014      0.017      0.127                                        0.005      0.005      0.021      0.030                                        0.011      0.011      0.006      0.002                                        0.143      0.183      0.010      0.009                                        0.057      0.056      0.011      0.009                                        0.204      0.217      0.011      0.013                                        0.033      0.040      0.009      0.014                                        0.064      0.062      0.008      0.009                                        0.105      0.118      0.004      0.005                                        0.014      0.016      0.011      0.007                                        0.489      0.524      0.008      0.044                                        0.507      0.594      0.017      0.061                                        0.279      0.318      0.013      0.028                                        5.360      6.110      13.060     18.590                                       9.820      11.190     15.620     19.340                                       7.500      9.280      14.620     19.020                                       1.292      1.855      3.820      14.400                                       0.146      0.179      0.440      3.070                                        0.006      0.006      0.008      0.008                                        __________________________________________________________________________

(9) Cloning

The hybridoma cell lines producing monoclonal antibodies as classifiedinto the above-mentioned five groups were subjected to cloning by thelimiting dilution to obtain a single clone. In performing the cloning,thymus cells prepared from BALB/c mice and suspended in HAT medium wasadded as the feeder cell at a rate of 1×10⁶ per well.

(10) Identification of Mouse Immunoglobulin Subclass

Identification of mouse immunoglobulin subclass was made with themonoclonal antibodies produced by the hybridoma cell lines which havebeen prepared as having a single clone by means of the above-mentionedcloning. The culture supernatants of the respective hybridoma cell lineswere assayed for the mouse immunoglobulin subclass identification usinga MONOAb typing kit (available from Zymed Co.)

The result was that seven clones have IgG1 type, eleven clones IgG2atype and three clones IgG2b type, with regard to the H chain. Regardingthe L chain, all the clones were found to have k chain (Table 1).

The respective clones classified into the five groups are shown in Table1.

EXAMPLE 2 Production of Monoclonal Antibodies

(1) Harvest of Ascitic Fluid

In order to obtain a higher concentration ofanti-human-cardiac-muscle-annexin-V monoclonal antibodies, the varioushybridoma cells are grown and the resulting respective clones wereinoculated, following washing three times with EMEM culture solution,into the ascitic cavities of BALB/c mice which have been administeredwith 2,6,10,14-tetramethyl pentadecane (pristane) in advance into theirascitic cavities. The ascitic fluids were harvested seven to fourteendays after the inoculation of the hybridoma cell lines into the asciticcavities. The ascitic fluids thus obtained were applied to centrifuge toremove the cellular and residual fractions. The supernatant fractionswere pooled for keeping at 4° C.

(2) Purification for the Anti-Human Cardiac-Muscle-Annexin-V MonoclonalAntibodies from the Ascitic Fluids

The respective anti-human-cardiac-muscle-annexin-V monoclonal antibodieswere purified by a combination of salting-out and ion-exchangechromatography to recover the purified IgG fractions.

Thus, 20 ml of each clone was added with an equal amount ofphosphate-buffered saline (PBS), followed by the addition of anhydroussodium sulfate to give a final concentration of 20%(w/v) with stirringat room temperature. The mixture was stirred for an additional one hour.

Centrifugation was then performed at 12,000 rpm with a high-speedcentrifuge (available form Hitachi. Co.) and the resulting residuefraction was dissolved in approx. 10 ml of phosphate-buffered saline.The solution was dialyzed three times against 20 mM sodium phosphatebuffer solution (pH 7.0) as the outer solution. On completing thedialysis, from the dialyzed solution the IgG fraction of theanti-human-cardiac-muscle-annexin-V monoclonal antibody was purified byion-exchange chromatography using a DEAE (diethylaminoethyl cellulose)column DE-52 (available from Whatman Co.) measuring 1.5 cm in innerdiameter and 8 cm in length equilibrated with 20 mM sodium phosphatebuffer solution (pH 7.0), from which the purified IIgG fraction wasobtained as the pass-through fraction or as the eluent fraction at aNaCl concentration in the range of 30 mM gradationally up to 50 mMformed with 20 mM sodium phosphate buffer solution (pH 7.0) containing30 mM to 50 mM NaCl. Each of the purified clones was determined for thepurity of IgG fraction, by high performance liquid chromatography usinga TSK gel G3000SW column (available from Tosoh. Co.) and sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using PhastSystemavailable from Pharmacia Co. The purity was found to be 95% or higher.

EXAMPLE 3 ELISA Assay System for Human Cardiac Muscle Annexin-V

(1) Preparation of Anti-Human-Cardiac-Muscle-Annexin-V MonoclonalAntibodies for HRPO-Labeling

The IgG fractions of clones HCA-155 and HCA-660HD were purified in themanner as described in Example 2 to study the application to ELISAsystem for human cardiac muscle annexin-V assay.

(2) Preparation of HRPO-Labeled Anti-Human-Cardiac-Muscle-Annexin-VMonoclonal Antibodies

(A) Preparation of Anti-Human-Cardiac-Muscle-Annexin-V MonoclonalAntibodies F(ab')₂

For labeling the IgG fractions of theanti-human-cardiac-muscle-annexin-V monoclonal antibodies, each 10 mg ofthe anti-human-cardiac-muscle-annexin-V monoclonal antibodies wassubjected to centrifugal filtration with a centrifugal concentrator(Centricon 10 available from Amicon Co.). to give a final volume of 1ml. The concentrated antibody was dialyzed against 0.1M sodium acetatebuffer (pH 4.0) containing 0.2M NaCl as the solvent.

The IgG solution resulting from the dialysis was added with a solutionof pepsin (available from Sigma Co.) dissolved in 1M sodium acetatebuffer (pH 4.0) containing 0.2M NaCl, so that the pepsin is 4% of theIgG content, followed by reaction at 37° C. for 6 to 16 hours. Oncompleting the reaction, the resultant was applied to molecular-sievechromatography using a Sephadex G-150 column (available from PharmaciaCo.) for gel filtration, which has been equilibrated with 0.1M sodiumborate buffer (pH 8.0), to obtain the F(ab')₂ fragments of therespective anti-human-cardiac-muscle-annexin-V monoclonal antibodies.

(B) Preparation of Anti-Human-Cardiac-Muscle-Annexin-V MonoclonalFab'-SH

Each of the anti-human-cardiac-muscle-annexin-V monoclonal antibodiesF(ab')₂ as prepared in the aforesaid (A) was subjected to centrifugationfor further concentration by a Centricon centrifugal concentrator toprepare the concentrated F(ab')₂ fractions of the respectiveanti-human-cardiac-muscle-annexin-V monoclonal antibodies.

The concentrated fraction was added with 0.1 ml of 100 mM2-mercaptoethylamine hydrochloride (available from Kishida ChemicalsCo.) solution for reaction at 37° C. for 90 minutes. On completing thereaction, the resultant was applied to a Sephadex G-25 column forequilibrium gel filtration (available from Pharmacia Co.) measuring 1.6cm in diameter and 20 cm in length which has been equilibrated with 0.1Msodium phosphate buffer (pH 6.0) containing 1 mM EDTA, for fractionalpurification of the Fab'-SH fraction, followed by centrifugalconcentration by a Centricon 10 centrifugal concentrator, to give afinal volume of 1 ml. Thus, the concentrated Fab'-SH fractions of clonesHCA-155H and HCA-660HD anti-human-cardiac-muscle-annexin-V monoclonalantibodies were prepared.

(C) Preparation of HRPO Maleimide

Ten mg (as a protein content) of HRPO (available from Boehlinger Co.)was dissolved in 1 ml of 0.1M sodium phosphate buffer (pH 6.0). Theresulting solution was added with 100 microliters solution ofN-hydroxysuccinimide ester (available from Zeeben Chemicals Co.)dissolved in dimethylformamide (DMF) (available from Kishida ChemicalsCo.) to give a final concentration of 25 mg/ml, followed by reaction at30° C. for 60 minutes to prepare maleimide ester of HRPO. On completingthe reaction, the solution was centrifuged for five minutes at 3000 rpm.The supernatant was applied to a Sephadex G-25 column for equilbrium gelfiltration (available from Pharmacia Co.) measuring 1.6 cm in diameterand 20 cm in length, which has been equilibrated with 0.1M sodiumphosphate buffer (pH 6.0), in order to purify HRPO maleimide. Thepurified fraction of HRPO maleimide was then subjected to centrifugalconcentration by a Centricon 10 centrifugal concentrator to prepare theconcentrated fraction of HRPO maleimide.

(D) Preparation of HRPO-Labeled Anti-Human-Cardiac-Muscle-Annexin-VMonoclonal Fab' Antibodies

The concentrated Fab'-SH fraction of eachanti-human-cardiac-muscle-annexin-V monoclonal antibody and theconcentrated fraction of HRPO maleimide were mixed together at a molarratio of 1:1, for reaction at 4° C. for 15 to 24 hours. On completingthe reaction, 2-mercaptoethylamine was added to give a concentration of2 mM in the reaction solution, followed by reaction at 37° C. for 20minutes in order to block unaltered HRPO maleimide. The resultant wasthen subjected to molecular-sieve gelchromatography using a UltragelACA44 column (available from Pharmacia Co.) equilibrated with 20 mMsodium phosphate--sodium citrate buffer containing 0.15M NaCl and 2.5 mMEDTA (pH 5.6) and measuring 1.6 cm in diameter and 65 cm in length, forremoving unaltered Fab'-SH of the anti-human-cardiac-muscle-annexin-Vmonoclonal antibodies and unaltered HRPO maleimide and purifying theHRPO-labeled anti-human-cardiac-muscle-annexin-V monoclonal Fab'antibodies (hereinafter referred to as HRPO-labeledanti-human-cardiac-muscle-annexin-V monoclonal antibodies).

(3) Preparation of Anti-Dog-32KP Polyclonal Antibodies IgG

Anti-dog-32KP-annexin-V polyclonal antiserum was obtained by immunizingrabbits with dog-derived purified 32KP annexin-V antigen. To three ml ofthe antiserum was added an equal amount of phosphate-buffered saline(PBS), followed by the addition of anhydrous sodium sulfate to give afinal concentration of 20% with stirring and an additional stirring forone hour at room temperature. The resultant was then centrifuged at12,000 rpm for 10 minutes, and the residue obtained was dissolved inapprox. 3 ml of saline. The solution was then subjected to dialysisagainst 20 mM sodium phosphate (pH 7.0) as the solvent. On completingthe dialysis, the resulting solution was applied to ion-exchangechromatography using a DEAE cellulose DE-52 column (available fromWhatman Co.) equilibrated with 20 mM sodium phosphate buffer (pH 7.0)and measuring 1.5 cm in diameter and 6 cm in length, to purify the IgGfraction of anti-dog-32KP-annexin-V polyclonal antibody. From 3 ml ofthe anti-dog-32KP-annexin-V polyclonal antibody-containing antiserumthere was obtained 13 mg of the purified IgG fraction.

(4) Preparation of HRPO-Labeled Anti-Dog-32KP-Annexin-V PolyclonalAntibody

(A) Preparation of Anti-Dog-32KP-Annexin-V Polyclonal Antibody F(ab')₂

For labeling the purified IgG fraction of the anti-dog-32KP-annexin-Vpolyclonal antibody, 12 mg of aforesaid antibody was applied to aCentricon 10 centrifugal concentrator (available from Amicon Co.) togive a concentrated volume of 1 ml, followed by dialysis against 0.1Msodium acetate buffer (pH 4.5) containing 0.2M NaCl.

The antibody solution resulting from the dialysis was added with asolution of pepsin (available from Sigma Co.) dissolved in 0.1M sodiumacetate (pH 4.5) containing 0.2M NaCl to give a concentration of 4%based on the IgG content, followed by reaction at 37° C. for 16 hours.On completing the reaction, the resultant was applied to molecular-sievechromatography using a Sephadex G-150 column for equilibrium gelfiltration (available from Pharmacia Co.) equilibrated with 0.1M sodiumborate buffer (pH 8.0) and measuring 1.6 cm in diameter and 65 cm inlength, to fractionate and purify the F(ab')₂ fraction of the antibody.The resulting fraction was subjected to dialysis against sodiumphosphate buffer (pH 6.0) containing 1 mM EDTA as the solvent. Oncompleting dialysis, the dialyzed solution was applied to centrifugalconcentration by a Centricon 10 centrifugal concentrator to give a finalvolume of 1 ml. The solution resulting from the dialysis was used toprepare the labeled antibody as the anti-dog-32KP-annexin-V F(ab')₂polyclonal antibody. Thus, from 12 mg of the IgG fraction of theanti-dog-32KP polyclonal antibody obtained in the above-mentionedmanner, there was prepared approx. 7 mg of the F(ab')₂ fraction.

(B) Preparation of Anti-Dog-32KP-Annexin-V Polyclonal Fab'-SH

To 7 mg/ml solution of anti-dog-32KP polyclonal antibody F(ab')₂fraction as prepared in above (A) was added 0.1 ml of 100 mM2-mercaptoethylamine chloride (available from Kishida Chemicals Co.) forreaction at 37° C. for 90 minutes. On completing the reaction, theresultant was applied to a Sephadex G-25 column (available fro PharmaciaCo.) for equilibrium gel filtration measuring 1.6 cm in diameter and 20cm in length equilibrated with 0.1M sodium phosphate buffer (pH 6.0)containing 1 mM EDTA, to purify the Fab'-SH fraction. The fraction wasthen subjected to centrifugal concentration by a Centricon 10centrifugal concentrator to a volume of 1 ml. Thus, from 7 mg of theF(ab')₂, there was obtained 6.1 mg of anti-dog32KP annexin-V polyclonalantibodies Fab'-SH.

(C) Preparation of HRPO Maleimide

Ten mg (as a protein content) of HRPO (available from Boehlinger CO.)was dissolved in 1 ml of 0.1M sodium phosphate buffer (pH 6.0). Theresulting solution was added with 100 microliters solution ofN-hydroxysuccinimide ester (available from Zeeben Chemicals Co.)dissolved in dimethylformamide (DMF) (available from Kishida ChemicalsCo.) to give a final concentration of 25 mg/ml, followed by reaction at30° C. for 60 minutes to prepare maleimide ester of HRPO. On completingthe reaction, the solution was centrifuged for five minutes at 3000 rpm.The supernatant was applied to a Sephadex G-25 column for equilibriumgel filtration (available from Pharmacia Co.) measuring 1.6 cm indiameter and 20 cm in length, which has been equilibrated with 0.1Msodium phosphate buffer (pH 6.0), in order to purify HRPO maleimide. Thepurified fraction of HRPO maleimide was then subjected to (centrifugal)concentration by a Centricon 10 centrifugal concentrator to prepare theconcentrated fraction of HRPO maleimide.

(D) Preparation of HRPO-Labeled Anti-Dog-32KP-Annexin-V PolyclonalAntibody Fab'

The Fab'-SH fraction of anti-dog-32KP-annexin-V polyclonal antibody thusobtained was mixed with the HRPO maleimide fraction at a molar ratio of1:1, for reaction at 4° C. for 15 to 24 hours. Then the resultant wasadded with 2-mercaptoethylamine hydrochloride to give a concentration of2 mM in the reaction solution, followed by reaction at 37° C. for 20minutes to block unaltered HRPO maleimide. The resultant was thensubjected to gelchromatography using a Ultrogel ACA44 column (availablefrom Pharmacia Co.) equilibrated with 20 mM sodium phosphate-sodiumcitrate buffer (pH 5.6) containing 0.15M NaCl and 2.5 mM EDTA andmeasuring 1.6 cm in diameter and 65 cm in length, for removing unalteredanti-dog-32KP-annexin-V polyclonal antibody Fab'-SH fraction andunaltered HRPO maleimide and purifying HRPO-labeledanti-dog-32KP-annexin-V polyclonal antibody (hereinafter referred to asHRPO-labelled antibodies).

(E) HRPO Activity Determination

In determining the HRPO enzyme activity of the HRPO-labeled antibodies,2.98 ml of 0.1M sodium phosphate buffer (pH 7.0) containing 0.2% phenol,0.5 mM hydrogen peroxide and 0.15 mg/ml 4-amino antipyrine were addedwith 20 microliters of the HRPO-labeled antibodies to give a totalvolume of 3.0 ml, followed by reaction at 37° C. for five minutes, andmeasurement of absorbance at 500 nm by means of the Rate assay. HRPOactivity was determined by measuring the difference in absorbance (ΔAbs)per one minute.

(5) Preparation of Anti-Human-Cardiac-Muscle-Annexin-V MonoclonalAntibodies Solid Phase

As monoclonal antibodies for use in the solid phase in an ELISA systemfor the assay of human cardiac muscle annexin-V clones HCA-290, HCA-627,HCA-57HDR, HCA-293HDR and HCA-350HDR were selected and the IgG fractionsof the respective clones were purified from the ascitic fluids in thesame manner described in Example 3.

The IgG fraction of each of the anti-human annexin-V monoclonalantibodies was adjusted to have a concentration of 30 micrograms/ml with0.1M sodium phosphate buffer (pH 7.5) containing 0.1% sodium azide, anddistributed in a microtiterplate for ELISA (available from Nunc Co.) ata rate of 100 microliters per well, for sensitization at 4° C.overnight. Then each well of the microtiterplate was washed three timeswith phosphate-buffered saline (PBS) containing 0.05% Tween 20surfactant, and then added with 300 microliters of PBS containing 1% BSA(as the blocking solution), followed by a further blocking operation at4° C. overnight, to prepare antibody-plates sensitized withanti-human-cardiac-muscle-annexin-V monoclonal antibodies (hereinafterreferred to as anti-human-annexin-V monoclonal antibody plates).

(6) Studies on ELISA Assay System Using Anti-Human-Annexin-V MonoclonalAntibodies

The anti-human-annexin-V monoclonal antibody plates prepared in theabove-mentioned manner were added, after the blocking solutiondiscarded, with 10 mM sodium phosphate buffer (pH 7.0) containing 1%BSA, 0.15M NaCl and 5 mM EDTA at a rate of 100 microliters per well.Standard antigen solutions were prepared, with 8M-urea-treated humanannexin-V antigen (obtained by the treatment with 8M urea), and nativeannexin-V (obtained by no treatment with 8M urea), to giveconcentrations of 1.5625 ng/ml, 3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25ng/ml, 50 ng/ml and 100 ng/ml. The respective standard antigen solutionswere added to the well at a rate of 20 microliters per well, followed bystirring and reaction for one hour at room temperature. On completingthe reaction, each well was washed three times with the washingsolution. To each of the washed wells were added the HRPO-labeledanti-human-annexin-V monoclonal antibodies Fab' (from clones HCA-155Hand HCA-660HD) at a rate of 100 microliters per well, for reaction atroom temperature for thirty minutes. Following the reaction, each wellwas washed six times with washing solution and then added with 100microliters of OPD substrate solution containing 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂, for reaction for thirty minutes. Thereaction was terminated by adding 2N H₂ SO₄ solution at a rate of 100microliters per well. Absorbance was determined on an ELISA plate readerby the dual wavelength method at 492 nm as the primary and at 690 nm asthe secondary.

The absorbance herein intended is obtained by subtracting an absorbancemeasurement at the secondary wavelength 690 nm from that at the primarywavelength 492 nm.

The results of the absorbance measured are given in Table 2 below, withrespect to 8M-urea-treated-human annexin-V standard antigen solutions.

                                      TABLE 2-1                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-15    5H                                                                            Antibody and HCA-290 Solid Phase (Absorbance A492 nm - 690 nm)                Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.006                                                                            0.010                                                                            0.080                                                                            0.042                                                                              -0.051                                                                            0.032                                                                              0.115                                      1.5625      0.043                                                                            0.048                                                                            0.041                                                                            0.004                                                                              0.037                                                                             0.044                                                                              0.051                                      3.125       0.093                                                                            0.090                                                                            0.084                                                                            0.005                                                                              0.080                                                                             0.089                                                                              0.098                                      6.25        0.192                                                                            0.160                                                                            0.195                                                                            0.009                                                                              0.144                                                                             0.182                                                                              0.221                                      12.5        0.352                                                                            0.340                                                                            0.357                                                                            0.009                                                                              0.332                                                                             0.350                                                                              0.367                                      25          0.738                                                                            0.736                                                                            0.726                                                                            0.006                                                                              0.720                                                                             0.733                                                                              0.746                                      50          1.582                                                                            1.618                                                                            1.615                                                                            0.020                                                                              1.565                                                                             1.605                                                                              1.645                                      100         2.990                                                                            2.894                                                                            2.892                                                                            0.056                                                                              2.813                                                                             2.925                                                                              3.037                                      __________________________________________________________________________

                                      TABLE 2-2                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-15    5H                                                                            Antibody and HCA-57HDR Solid Phase (Absorbance A492 nm - 690 nm)              Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.007                                                                            0.007                                                                            0.005                                                                            0.001                                                                              0.004                                                                             0.006                                                                              0.009                                      1.5625      0.042                                                                            0.039                                                                            0.041                                                                            0.002                                                                              0.038                                                                             0.041                                                                              0.044                                      3.125       0.079                                                                            0.093                                                                            0.091                                                                            0.008                                                                              0.073                                                                             0.088                                                                              0.103                                      6.25        0.193                                                                            0.200                                                                            0.187                                                                            0.007                                                                              0.180                                                                             0.193                                                                              0.206                                      12.5        0.311                                                                            0.383                                                                            0.431                                                                            0.060                                                                              0.254                                                                             0.375                                                                              0.496                                      25          0.889                                                                            0.704                                                                            0.785                                                                            0.093                                                                              0.607                                                                             0.793                                                                              0.978                                      50          1.631                                                                            1.393                                                                            1.642                                                                            0.141                                                                              1.274                                                                             1.555                                                                              1.837                                      100         2.788                                                                            2.720                                                                            2.540                                                                            0.128                                                                              2.426                                                                             2.683                                                                              2.939                                      __________________________________________________________________________

                                      TABLE 2-3                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-15    5H                                                                            Antibody and HCA-293HDR Solid Phase (Absorbance A492 nm - 690 nm)             Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.004                                                                            0.009                                                                            0.007                                                                            0.003                                                                              0.002                                                                             0.007                                                                              0.012                                      1.5625      0.064                                                                            0.067                                                                            0.048                                                                            0.010                                                                              0.039                                                                             0.060                                                                              0.080                                      3.125       0.090                                                                            0.097                                                                            0.110                                                                            0.010                                                                              0.079                                                                             0.099                                                                              0.119                                      6.25        0.188                                                                            0.190                                                                            0.190                                                                            0.001                                                                              0.187                                                                             0.189                                                                              0.192                                      12.5        0.395                                                                            0.399                                                                            0.416                                                                            0.011                                                                              0.381                                                                             0.403                                                                              0.426                                      25          0.771                                                                            0.825                                                                            0.869                                                                            0.049                                                                              0.723                                                                             0.822                                                                              0.920                                      50          1.781                                                                            1.783                                                                            1.811                                                                            0.017                                                                              1.758                                                                             1.792                                                                              1.825                                      100         3.470                                                                            3.740                                                                            3.700                                                                            0.146                                                                              3.345                                                                             3.637                                                                              3.928                                      __________________________________________________________________________

                                      TABLE 2-4                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-15    5H                                                                            Antibody and HCA-350HDR Solid Phase (Absorbance A492 nm - 690 nm)             Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.006                                                                            0.006                                                                            0.006                                                                            0.000                                                                              0.006                                                                             0.006                                                                              0.006                                      1.5625      0.059                                                                            0.059                                                                            0.059                                                                            0.000                                                                              0.059                                                                             0.059                                                                              0.059                                      3.125       0.113                                                                            0.128                                                                            0.116                                                                            0.008                                                                              0.103                                                                             0.119                                                                              0.135                                      6.25        0.184                                                                            0.178                                                                            0.215                                                                            0.020                                                                              0.153                                                                             0.192                                                                              0.232                                      12.5        0.467                                                                            0.572                                                                            0.585                                                                            0.065                                                                              0.412                                                                             0.541                                                                              0.671                                      25          0.954                                                                            0.840                                                                            1.062                                                                            0.111                                                                              0.730                                                                             0.952                                                                              1.174                                      50          2.313                                                                            2.259                                                                            2.042                                                                            0.143                                                                              1.918                                                                             2.205                                                                              2.492                                      100         3.280                                                                            3.360                                                                            3.200                                                                            0.080                                                                              3.120                                                                             3.280                                                                              3.440                                      __________________________________________________________________________

                                      TABLE 2-5                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-66    0HD                                                                           Antibody and HCA-290 Solid Phase (Absorbance A492 nm - 690 nm)                Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.007                                                                            0.007                                                                            0.008                                                                            0.001                                                                              0.006                                                                             0.007                                                                              0.008                                      1.5625      0.052                                                                            0.052                                                                            0.053                                                                            0.001                                                                              0.051                                                                             0.052                                                                              0.053                                      3.125       0.121                                                                            0.116                                                                            0.107                                                                            0.007                                                                              0.100                                                                             0.115                                                                              0.129                                      6.25        0.257                                                                            0.243                                                                            0.211                                                                            0.024                                                                              0.190                                                                             0.237                                                                              0.284                                      12.5        0.497                                                                            0.427                                                                            0.494                                                                            0.040                                                                              0.394                                                                             0.473                                                                              0.552                                      25          0.950                                                                            0.977                                                                            0.842                                                                            0.071                                                                              0.780                                                                             0.923                                                                              1.066                                      50          2.142                                                                            2.124                                                                            2.038                                                                            0.056                                                                              1.990                                                                             2.101                                                                              2.212                                      100         3.980                                                                            3.590                                                                            3.810                                                                            0.196                                                                              3.402                                                                             3.793                                                                              4.184                                      __________________________________________________________________________

                                      TABLE 2-6                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-66    0HD                                                                           Antibody and HCA-57HDR Solid Phase (Absorbance A492 nm - 690 nm)              Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.007                                                                            0.008                                                                            0.008                                                                            0.001                                                                              0.007                                                                             0.008                                                                              0.009                                      1.5625      0.058                                                                            0.056                                                                            0.048                                                                            0.005                                                                              0.043                                                                             0.054                                                                              0.065                                      3.125       0.108                                                                            0.113                                                                            0.113                                                                            0.003                                                                              0.106                                                                             0.111                                                                              0.117                                      6.25        0.294                                                                            0.228                                                                            0.309                                                                            0.043                                                                              0.191                                                                             0.277                                                                              0.363                                      12.5        0.448                                                                            0.544                                                                            0.461                                                                            0.052                                                                              0.380                                                                             0.484                                                                              0.588                                      25          0.907                                                                            0.884                                                                            0.870                                                                            0.019                                                                              0.850                                                                             0.887                                                                              0.924                                      50          1.862                                                                            1.813                                                                            1.774                                                                            0.044                                                                              1.728                                                                             1.816                                                                              1.905                                      100         3.440                                                                            3.430                                                                            3.600                                                                            0.095                                                                              3.299                                                                             3.490                                                                              3.681                                      __________________________________________________________________________

                                      TABLE 2-7                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-66    0HD                                                                           Antibody and HCA-293HDR Solid Phase (Absorbance A492 nm - 690 nm)             Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.006                                                                            0.007                                                                            0.009                                                                            0.002                                                                              0.004                                                                             0.007                                                                              0.010                                      1.5625      0.076                                                                            0.074                                                                            0.086                                                                            0.006                                                                              0.066                                                                             0.079                                                                              0.092                                      3.125       0.157                                                                            0.117                                                                            0.114                                                                            0.024                                                                              0.081                                                                             0.129                                                                              0.177                                      6.25        0.233                                                                            0.223                                                                            0.247                                                                            0.012                                                                              0.210                                                                             0.234                                                                              0.258                                      12.5        0.516                                                                            0.492                                                                            0.490                                                                            0.014                                                                              0.470                                                                             0.499                                                                              0.528                                      25          1.030                                                                            0.927                                                                            1.056                                                                            0.068                                                                              0.868                                                                             1.004                                                                              1.141                                      50          2.042                                                                            2.535                                                                            2.167                                                                            0.256                                                                              1.735                                                                             2.248                                                                              2.761                                      100         4.010                                                                            4.060                                                                            4.230                                                                            0.115                                                                              3.869                                                                             4.100                                                                              4.331                                      __________________________________________________________________________

                                      TABLE 2-8                                   __________________________________________________________________________    Assay of 8M-urea-treated Human Annexin-V Antigen by Monoclonal Antibodies     ELISA                                                                         System                                                                        Combination of Monoclonal Antibodies for ELISA System: HRPO-labeled-HCA-66    0HD                                                                           Antibody and HCA-350HDR Solid Phase (Absorbance A492 nm - 690 nm)             Concentration of 8M-                                                                      Number of                                                                              Standard                                                 urea-treated Human                                                                        Measurements                                                                           Deviation                                                                          Average                                                                           Average                                         Annexin-V Antigen (ng/ml)                                                                 1  2  3  (SD) -2SD                                                                              Average                                                                            +2SD                                       __________________________________________________________________________    0           0.007                                                                            0.008                                                                            0.009                                                                            0.001                                                                              0.006                                                                             0.008                                                                              0.0.010                                    1.5625      0.079                                                                            0.067                                                                            0.052                                                                            0.014                                                                              0.039                                                                             0.066                                                                              0.093                                      3.125       0.146                                                                            0.164                                                                            0.098                                                                            0.034                                                                              0.068                                                                             0.136                                                                              0.204                                      6.25        0.254                                                                            0.293                                                                            0.340                                                                            0.043                                                                              0.210                                                                             0.296                                                                              0.382                                      12.5        0.532                                                                            0.530                                                                            0.548                                                                            0.010                                                                              0.517                                                                             0.537                                                                              0.556                                      25          0.976                                                                            1.007                                                                            1.138                                                                            0.086                                                                              0.868                                                                             1.040                                                                              1.212                                      50          2.000                                                                            2.111                                                                            1.998                                                                            0.065                                                                              1.907                                                                             2.036                                                                              2.166                                      100         3.650                                                                            3.450                                                                            3.400                                                                            0.132                                                                              3.235                                                                             3.500                                                                              3.765                                      __________________________________________________________________________

FIGS. 1 thorough 8 illustrate calibration curves for human annexin-V, inwhich the results of measurement as given in Tables 2-1 through 2-8 areplotted in diagrams with the absiccia being the concentration of humanannexin-V antigen and the ordinate being the difference in theabsorbance measured between at 492 nm and at 690 nm.

The calibration curves are excellent ones as the absorbance increaseswith increasing concentration of human annexin-V substantially linearlyas high as a concentration of 100 ng/ml. The use of these calibrationcurves enables reliable assay of human annexin-V concentration insamples to be examined (plasma and serum). It is also possible todetermine the concentration of human annexin-V with good reproducibilityeven as low as a concentration of 1 ng/ml, as can be seen FIG. 1. Thecalibration curves as shown by FIGS. 1 through 8 are of reliable use forthe determination of the concentration of human annexin-V.

With the native human annexin-V without 8M-urea-treatment as thestandard antigen, the absorbance detected was approx. 5% to 10% of thatwith 8M-urea-treated human annexin-V, demonstrating that the system hasa low reactivity to the native human annexin-V antigen.

EXAMPLE 4 Assay of Human Annexin-V Using Anti-Human-Annexin-V MonoclonalAntibody HCA-627 and HRPO-Labeled-Anti-Dog-32KP-Annexin-V PolyclonalAntibody

Assay of human annexin-V concentration was conducted by ELISA usinganti-human-annexin-V monoclonal antibody HCA-627, which has been foundto exhibit a high reactivity with native annexin-V without 8M-ureatreatment, in combination with anti-dog-32KP-annexin-V polyclonalantibody.

Thus, 100 microliters of anti-human-annexin-V monoclonal antibodyHCA-627, secreted from hybridoma cell line HCA-627 strain capable ofproducing an anti-human-annexin-V monoclonal antibody, was distributedin wells at a rate of 100 microliters per well, to prepare solid-phasewell coupled with anti-human-annexin-V monoclonal antibody in the mannerfor preparing the solid-phase as described in Example 4 (5).

To the solid phase wells was added 10 mM sodium phosphate buffer (pH7.0) containing 1% BSA, 0.15M NaCl and 5 mM EDTA at a rate 100microliters per well.

Following the distribution of the reaction buffer, each well was addedwith a standard solution. As the standard solution in this Example wereused standard solutions of 8M-urea treated human annexin-V antigen andstandard solutions of native human annexin-V antigen. Thus, one set ofthe solid phase wells were added with the standard solution of8M-urea-treated human annexin-V whereas the other set of the solid phasewells were added with the standard solutions of native annexin-Vantigen. Following the distribution of the standard solutions, anantigen-antibody reaction was allowed to take place in each well withstirring for one hour. After the antigen-antibody reaction, each wellwas washed four times with the washing solution and then added with 100microliters of the HRPO-labeled-anti-dog-annexin-V polyclonal Fab'antibody (100 mU per ml of the reaction buffer) followed by anantigen-antibody reaction for thirty minutes with stirring. Then, eachwell was washed eight times with the washing solution and added with 100microliters of OPD substrate solution for the color production reactionat room temperature for thirty minutes.

With the lapse of the time for the color production, each well was addedwith the reaction-terminating solution (2N H₂ SO₄ solution) to terminatethe reaction, followed by absorbance measurements at the primarywavelength 492 nm and secondary wavelength 690 nm on an ELISA platereader to assay the samples and the standard solutions for theconcentration of human annexin-V.

The absorbance was obtained by subtracting an absorbance measured at thesecondary wavelength 690 nm from that at the primary wavelength 492 nm.Thus, a calibration curve was prepared by plotting the absorbance dataagainst the human annexin-V concentration with reference to the antigenprotein standard solution. Based on such calibration curve, a sample canbe calibrated for the concentration of the antigenic protein, humanannexin-V.

In the Table 3 below, there are given absorbance data on the standardantigen of native annexin-V obtained by ELISA using anti-human annexin-Vmonoclonal antibody HCA-627 and the HRPO-labeled-anti-dog-32KPpolyclonal antibody.

                  TABLE 3                                                         ______________________________________                                        Combination System in Assay of Native Annexin-V Antigen by ELISA              Combination of HRPO-labeled anti-dog-32KP Polyclonal Antibody                 and Anti-human-annexin-V Monoclonal Antibody HCA-627 Solid                    Phase (Absorbance = A492 nm - 690 nm)                                         Concentration of                                                                         Number of  Standard            Aver-                               Native Annexin-V                                                                         Measurements                                                                             Deviation                                                                              Average                                                                             Aver-                                                                              age                                 Antigen (ng/ml)                                                                          1      2       (SD)   -25D  age  +2SD                              ______________________________________                                        0          0.018  0.019   0.001  0.017 0.019                                                                              0.020                             1.5625     0.037  0.038   0.001  0.036 0.038                                                                              0.039                             3.125      0.061  0.063   0.001  0.059 0.062                                                                              0.065                             6.25       0.119  0.122   0.002  0.116 0.121                                                                              0.125                             12.5       0.215  0.218   0.002  0.212 0.217                                                                              0.221                             25         0.421  0.433   0.008  0.410 0.427                                                                              0.444                             50         0.825  0.826   0.001  0.824 0.826                                                                              0.827                             100        1.448  1.512   0.045  1.389 1.480                                                                              1.571                             ______________________________________                                    

FIG. 9 illustrates a calibration curve prepared from the results of theassay as given in Table 3. Thus, the reliable calibration curve wasobtained for the concentration of the native human annexin-V antigen inthe ELISA system using the anti-human-annexin-V monoclonal antibodyHCA-627 and the HRPO-labeled anti-dog-32KP polyclonal antibody.

EXAMPLE 5 Detection and Quantitative Analysis of Human Annexin-V inSamples from Patients with Myocardial Infarction,by ELISA UsingAnti-Human-Annexin-V Monoclonal Antibody HCA-627 andHRPO-Labeled-Anti-Dog-32KP Polyclonal Fab'

The combination of anti-human-annexin-V monoclonal antibody HCA-627 andHRPO-labeled-anti-dog-32KP polyclonal Fab' prepared in theabove-mentioned manner, which has been found to have an excellentsensitivity in assaying the native human annexin-V, was examined for itsapplicability to the assay of human annexin-V concentration in theclinical samples from patients with myocardial infarction.

Thus, the solid phase composed of anti-human-annexin-V monoclonalantibody was added, after disharging the solution, with 10 mM sodiumphosphate (pH 7.0) containing 1% BSA, 0.15M NaCl and 5 mM EDTA at a rateof 100 microliters per well. Antigen standard solutions of native humanannexin-V (1.5625 ng/ml, 3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml,50 ng/ml and 100 ng/ml) and human plasma samples are added each at arate of 20 microliters per well, followed by stirring and anantigen-antibody reaction for one hour at room temperature. With thelapse of the time for the antigen-antibody reaction, each well waswashed three times with the washing solution. Then, to the wells wasadded HRPO-labeled-anti-dog-32KP polyclonal Fab' antibody having beenprepared to have an appropriate concentration (100 mU per ml of thereaction buffer) at a rate of 100 microliters per well, followed by anantigen-antibody reaction for thirty minutes at room temperature. Withthe lapse of the time for the antigen-antibody reaction, each well waswashed eight times with the washing solution.

Following the washing, each well was added with 100 microliters of OPDsubstrate solution containing 0.1 m phosphate-citrate buffer, 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂, for color production reaction forthirty minutes at room temperature.

With the lapse of the time for the color production reaction, each wellwas added with 2N H₂ SO₄ as reaction-terminating solution to terminatethe color production reaction. The color produced in each well wasassayed at the primary wavelength 492 nm and at the secondary wavelength690 nm. The assay was conducted by measuring absorbance on an ELISAplate reader at the primary wavelength 492 nm and at the secondarywavelength 690 nm. The absorbance for a sample to be examined or for astandard solution was obtained by subtracting an absorbance measurementat the secondary wavelength 492 nm from that at the primary wavelength690 nm, to determine the concentration of human cardiac-muscle annexin-Vin the sample or the standard solution.

The measurements for the human plasma samples were obtained by readingthe human annexin-V concentration in the respective samples withreference to the calibration curve which has been prepared based on theabsorbance measurements of the standard solutions of varying humanannexin-V concentration.

EXAMPLE 6 Application of the Enzyme-Antibody Method of the Invention tothe Assay of Blood Samples

(1) Diagnosis of Myocardial Infarction

A 75-year-old man with a pain in the chest at 6:00 a.m. was hospitalizedat 8:30 a.m. of the same day on suspicion of acute myocardial infarctionbecause of abnormality on the electrocardiogram and the pain in thechest. At 9:00 a.m. of the day (i.e. three hours from the the attack)while CK (creatine phosphate enzyme) value indicated 108 U/L in thenormal range, annexin-V was found to be an increased value of 90.4ng/ml. With the lapse of three days from attack, annexin-V decreaseddown to 13.1 ng/ml. The abnormality on the electrocardiogram in thiscase indicated the finding of acute myocardial infarction and the assayof human annexin-V concentration in the plasma sample was found to be ofuse in a quick diagonsis of myocardial infarction.

It was later established that 70% of patients are diagnostic ofmyocardial infarction, in cases where the annexin-v concentrations inthe plasma of the patients were determined to be 50 ng/ml or more as theannexin-V concentration in the blood of the patient with myocardialinfarction is relatively high.

(2) Diagnosis of Angina Pectoris

An 58-year-old man, who complained of two times of pains in the chestattacked at 2:30 p.m. and 6:30 p.m. and continued over 10 to 15 minutes,was hospitalized at 7:30 of the same day on suspicion of angina pectorisbecause of abnormality in the electrocardiogram and the relatively shortpain in the chest. While the CK value was a normal value of 83 U/L, theannexin-V concentration was an increased value of 29.9 ng/ml. Theannexin-V value decreased down to 29.9 ng/ml at 12:00 and 15.1 ng/ml at15:00 of the same day, demonstrating the restoration to normal. The casewas assessed to be suspicions of angina pectoris because of the clinicalsymptom and the abnormality on the electrocardiodiagram was thendiagnostic of angina pectoris, in which it was found that theconcentration of human annexin-V in the plasma also increases in a caseof angina pectoris.

The concentration of human annexin-V in the blood of a patient withangina pectoris is much higher than the normal value, although it isrelatively low as compared with in the case of myocardial infarction. Itwas later established that 70% of the patients were diagnostic of anginapectoris, in cases where the human annexin-V concentrations in theplasma of the patients were determined to be 20 to 50 ng/ml.

(3) Annexin-V Concentration in the Blood from Normal Adults

Annexin-V concentration in the blood from normal male adults 24 to 76years of age were determined to be 0.9 to 11.3 ng/ml, whereas those fromnormal female adults 24 to 76 years of age were 0.8 to 10.6 ng/ml, withthe average being 6.2 ng/ml, the maximum 11.3 ng/ml and the minimum 0.8ng/ml. However, there were observed no age-dependent variations.

EXAMPLE 7 Application of the Enzyme Antibody Method to Cardiac MuscleTissue Specific Staining

(1) The anti-human-annexin-V monoclonal antibodies obtained in Example 3was applicable not only to an ELISA method but to staining specificallylocalized or distributed areas of human annexin-V in the cardiac muscletissue by means of an indirect enzyme antibody method.

Furthermore, the use of the HRPO-labeled anti-human-annexin-V antibodiesprepared in Example 4 makes it possible to make a short-time specificstaining of the cardiac muscle tissue by means of a direct enzymeantibody method.

The following is given by taking the indirect enzyme antibody method asan example: paraffin-treated or frozen slices of the cardiac muscletissue were fixed on a slide glass and allowed, following theparaffin-removal or blocking, to react with the anti-human annexin-Vmonoclonal antibodies having been prepared to have an appropriateconcentration (IgG concentration: 500 to 1000 micrograms/ml) as theprimary antibody at room temperature over an hour in a humid box. Theresultant was then washed with phosplate-buffered saline (PBS) threetimes for five minutes each, followed by a reaction with HRPO-labeledanti-mouse-immunoglobulin antibodies as the secondary antibody forthirty minutes in a humid box. The resultant was washed three times forfive minutes each, followed by addition of substrate solution of4-aminoantipyrine type or diaminobenzidine for reaction over thirtyminutes. On completing reaction, the slide-glass was washed with PBS andthen sealed with a sealant such as glycerol through a covering glass,for a microscopic observation of the stained areas.

The results are given in FIGS. 10 and 11, in which the distribution oflocalized human annexin-V in the cardiac muscle tissue was stained withbrown color and developed in black, demonstrating that the invention isapplicable to a tissue-staining for cardiac muscle by means of an enzymeantibody method as well as to an ELISA.

EXAMPLE 8 Hybridoma Preparation

(1) Mice

Inbred-strain BALB/c female mice 5 to 8 weeks of age were maintained ona diet of the standard pellets, feeding water ad libitum in ananimal-breeding chamber (23°+1° C., humidity 70%).

(2) Inmmunization

Dog-annexin-V extracted and purified from dog cardiac muscles (cf.Japanese Laid-open patent application No.72147/1995) and the humanannexin-V purified from human cardiac muscles as described in Example 1were each dissolved in 0.1M sodium phosphate buffer (pH 7.6) anddialyzed against the same buffer. Following the dialysis, the respectiveannexin-V antigens was prepared so as to give solutions of aconcentration of 1 mg/ml, which were distributed into Eppen tubes at arate of 50 microliters per tube, followed by freeze-drying at -400° C.to keep the purified native dog- and human-cardiac muscle-derivedannexin-V antigens for immunization.

Dog annexin-V 0.2 ml (200 micrograms) as prepared in the above-mentionedmanner was added with 0.3 ml of physiological saline to give a volume of0.5 ml. The antigen solution thus prepared was mixed with 0.5 ml ofFreund's complete adjuvant, followed by thorough emulsification. The dogannexin-V in emulsion as the antigen was administered into the abdominalcavities of four female mice of 5 week-of age in a dose of the purifieddog annexin-V 40 micrograms per mouse for the intial immunization.

Dog annexin-V antigen 0.2 ml (200 micrograms) as prepared in theaforesaid manner was taken in a test tube, to which was added 0.3 ml ofphysiological saline to give a volume of 0.5 ml. The solution thusprepared was added with 0.5 ml of RIBI Adjuvant System (RAS) MPL+TDMEumulsion R-700 (available from RIBI. Immuno Chem. Research Inc.,Hamilton, Mont., USA), in which 1 ml of plysiological saline was addedper vial for making the solution. The resultant mixture was vigorouslystirred in a vortex mixer for approx. three minutes for emulsification.With this dog annexin-V antigen in emulsion the mice were immunized asbooster immunizations two weeks, four weeks and six weeks after theintial immunization in a dose of 50 maicrograms per mouse. A furtherbooster immunization was carried out twenty-three weeks after theinitial immunization in a dose of the dog annexin-V antigen 30micrograms per mouse, with the antigen having been prepared in the samemanner as mentioned above using RIBI Adjuvant System (RAS) MPL+TDMEmulsion, R-700. Following the booster immunization the blood wascollected from the respective mice at intervals to prepare sera, whichwere assayed by the aforesaid absorption test for antibody titersagainst native dog cardiac muscle- and human-annexin-V to examine theproduction of specific antibodies. Based on the result of the assay forthe antibody titer, mice were selected having a high titer. The selectedmice were administered, twenty-six weeks after the initial immunization,with the native annexin-V (having been prepared by dissolving in 1 ml ofphysiological saline to give a concentration of 50 micrograms/ml)intravenovsly through the mice tails as a booster. At this point themouse sera were again assayed for the antibody titer to select micehaving a higher titer. The selected mice were administered, one weekafter the preceding booster immunization, with 1 ml of the native humanannexin-V (having been prepared with physiological saline to give aconcentration of 100 micrograms/ml) slowly and intravenously through thetails of the mice for final immunization.

(3) Preparation of Spleen Cells

Three days after the final immunization, the spleen were excised fromthe BALB/c mice in the same manner as described in Example 1. Theexcised spleen cells were suspended in EMEM culture medium (availablefrom Nissui Co.) to prepare a suspension of spleen cells. The spleencells were washed four times with EMEM culture medium and determined forthe number of cells. The resultant spleen cells are 4.7×10⁸.

(4) Preparation of Parent Strain

Cell fusion was conducted using P3-X63-Ag8.653 culture cells(hereinafter referred to as X63 cells) as parent cell strain, which are2-amino-6-oxy-8-azapurine (8-Azaguanine) -resistant and derived fromBALB/c mouse myeloma.

X63 cells in the logarithmic growth phase were employed to subculture inRPMI-1640 medium (in a concentration of 20 micrograms/ml and with8-azaguanine) (available from GIBCO Co.) containing 10% of immobilizedfatal calf serum (hereinafter referred to as FCS). Further cultivationwas carried out, from three days in advance to the cell fusion, inRPMI-1640 culture medium containing 10% FCS but not containing8-azaguanine to maintain the growth ability in the logarithmic phase.The X 63 cells were washed three times with RPMI-1640 medium anddetermined for the number of cells. The number of living X63 cells was1.5×10⁸.

(5) Cell Fusion

Polyethylene glycol-400 available from Sigma Co. was dissolved inRPMI-1640 culture medium to give a concentration of 50% (W/V), followedby heating at 37° C. for use.

The spleen cells and X63 cells were mixed together in a ratio of thespleen cells: the X63 cells=10:1. The mixture was centrifuged at 1500rpm for five minutes, the supernatant was removed and the pelletizedcells were thoroughly disintegrated for use in cell fusion. The cellfusion was conducted, using polyethylene glycol prepared in theaforesaid manner and kept at 37° C., in accordance with the methoddescribed in "Kohler and Milstein: Nature, Vol.256, pages 495-497(1975)" and "European Journal of Immunology, Vol.6, pages 511-519(1976)".

The cell line following the cell fusion were suspended in HAT selectionmedium containing RPMI-1640 added with FCS of a 10% concentration,1×10⁻⁴ hypoxanthine, 4×10⁻⁷ aminopterin and 1.6×10⁻⁵ M thymidine, togive a spleen-cell concentration of 2.0×10⁶ per ml. The cell suspensionwas distributed in a 96-well microtitreplate at a rate of 50 microlitersper well, and incubated in an incubator kept at a 37° C. temperature anda 95% humidity under a 8% CO₂ atomsphere. One drop of the HAT medium wasadded to each well on the first day and the second day from the start ofthe incubation, followed by the addition of two drops of the same mediumseven and nine days after start, for further incubation. Then,incubation was carried out in a HAT-free culture medium. Appox. ten daysto two weeks after the start of incubation, screening was made, as thefirst screening, for clones producing anti-annexin-V monoclonalantibodies having a common reactivity to native human annexin-V andnative dog annexin-V, in which the screening was done by an annexin-Vabsorption test using the native human annexin-V and dog annexin-Vantigens.

The selected clones were then subjected to the second screening by meansof a capture method for monoclonal antibodies, through which wereselected hybridoma cell line clones producing monoclonal antibodiessuitable for use in a sandwich ELISA system for a sensitive assay ofannexin-V in combination with monoclonal antibodies HCA-627. Themonoclonal antibodies HCA-627 are produced by hybridoma cell lineHCA-627 strain which has been deposited under the number BP-5284 at theNational Institute of Bioscience and Human-Technology, the Agency ofIndustrial Aeience and Technology of Japan.

(6) Screening

(i) First Screening

As cell clones appeared ten days after the start of incubation, theabsorption test with native dog- and human-annexin-V as the antigens wascarried out by ELISA for the supernatants of the hybridoma cell linecultures.

It was found by the present inventors that an ELISA system employing acombination the monoclonal antibodies HCA-627 bound to the cells as thesolid phase and the HRPO-labeled anti-dog-32KP polyclonal antibodies canbe applied to the assay of native human annexin-V and dog annexin-Vantigens. The system was thus utilized to screen monoclonal antibodieswhich will efficiently react with the native annexin-V.

Thus, the first set was constituted of the supernatant of hybridoma cellline clone culture obtained by the subject cell fusion and the nativehuman annexin-V antigen solution (100 ng/ml) with each being distributedin U-bottomed wells of a microtitreplate at a rate of 50 microliters perwell, while the second set was constituted of the supernatant of thesame hybridoma cell line clone culture and the native dog annexin-Vantigen solution (100 ng/ml) with each being distributed in a pluralityof U-bottomed wells of another microtitreplate at a rate of 50microliters per well.

Each set was added with 50 microliters of 20% suspension of Sepharose 4Bcombined with anti-mouse-immunoglobulin antibody and allowed to standfor 10 minutes following stirring for the one hour. With each set, whenit was observed that the anti-mouse-immunoglobulin antibody-combinedsepharose 4B completely sedimented on the well-bottoms, the respectivesupernatants 50 microtilers were assayed for human annexin-V or dogannexin-V content remaining therein by means of the aforesaid annexin-VELISA method.

(ii) Selection of Monoclonal Antibodies by the First Screening

In this assay, if any anti-cardiac-muscle-annexin-V monoclonalantibodies against native dog- and human-annexin-V remain in the culturesupernatant of a hybridoma cell line, a reaction will occur between thenative dog- or human-annexin-V and the anti-human-annexin-V monoclonalantibodies, followed by a reaction with anti-mouse-immunoglobulinmonoclonal antibody-combined Sepharose 4B, resulting in the formation ofantigen-antibody complexes which are to sediment. This will lead to adecrease in the annexin-V content remaining in the supernatant and it istherefore possible to identify the occurrence of any anti-annexin-Vmonoclonal antibodies.

(iii) Second Screening

If there were successfully selected anti-annexin-V monoclonal antibodieshaving a reactivity with native human- and dog-annexin-V antigens,screening was then made, through the following capture ELISA, forhybridoma cell line clones producing any monoclonal antibodies suitablefor establishing a sandwich ELISA system for sensitive assay ofannexin-V, in combination with the monoclonal antibodies HCA-627produced by hybridoma cell line HCA-627 strain which has been depositedas an International Deposit under the number FERM BP-5284.

(iv) Capture ELISA Method

A solution was prepared of the IgG fraction of polyclonal antibodyhaving a specificity to the Fc portion of goat-derived anti-mouseimmunoglobulin (available from Nordic Co.), with 0.1M sodium phosphatesolution (pH 7.5) containing 0.1% sodium azide to give a concentrationof 50 microliters/ml. The solution was distributed in a 96-welltiterplate (available from Nunk Co,) at a rate of 50 microliters perwell for adsorption at 4° C. overnight.

The resultant was then washed three times with washing phosphate buffer(hereinafter referred to washing solution) containing 0.05% Tween-20,followed by blocking with PBS containing 1% BSA to prepareanti-mouse-immunoglobulin G-Fc polyclonal antibody-bound wells as thesolid phase.

(v) Based on the results of the absorption test with native human- anddog-annexin-V, the culture supernatants of hybridoma cell linesproducing monoclonal antibodies with a high reactivity with the nativehuman- and dog annexin-V was added into the wells bound with theanti-mouse-immunoglobulin G-Fc polyclonal antibody as the solid phase ata rate 50 microliters per well, following the removal of the blockingsolution from each well. Upon the completion of reaction for one hour atroom temperature, each well was washed three times with the washingsolution. With a phosphate buffer of pH 7.0 containing 1% BSA and 5 mMEDTA (hereinafter reaction buffer solution), a solution of native humanannexin-V was prepared to give a concentration of 100 ng/ml. To eachwell was added 50 microliters of the solution of native human annexin-V,for a further reaction over one hour, followed by washing three timeswith the washing solution. HRPO-labeled HCA-627-Fab' antibody wereconditioned with the reaction buffer solution to have an appropriateconcentration, and then added to the respective wells for reaction overthirty minutes. The dilution of the HRPO-labeled antibodies was done insuch manner that the mouse immunoglobulin was 10 micrograms/ml, in orderto block the adsorption of the HRPO-labeled antibodies onto the wellbound with the anti-mouse -immunoglobulin G-Fc polyclonal antibodies asthe solid phate. On completing the reaction, each well was washed threetimes with the washing solution, followed by reaction with OPD substratesolution (containing 0.1M phosphate-citrate buffer added with 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂) for five minutes at room temperature.The reaction was terminated with 2N H₂ SO₄, to determine absorbance atthe primary wavelength 492 nm and at the secondary wavelength 690 nm onan ELISA plate reader. Measurements at the primary wavelength 492 nm areattributed to the substrate chromogen produced by the boundlabeled-antibody, whereas the measurements at the secondary wavelengthreflect flaws or stains on the microtiterplate.

By subtracting an absorbance measurement at the secondary wavelengthfrom an absorbance measurement at the primary wavelength, it is possibleto determine the true absorbance in response to the amount of chromogenchanged due to the enzyme on the labeled-antibodies which have beenbound to antigen in proportion to the amount of the antigen bound to thesolid-phase.

This second screening enables obtainment of an annexin-V monoclonalantibody having reactivities with the native human- and dog-annexin-Vantigens and being capable of recognizing an antigen determinant sitedifferent from another site on the indentical annexin-V molecule to berecognized by anti-annexin-V monoclonal antibody HCA-627, therebyproviding an optimal combination with the anti-annexin-V monoclonalantibody HCA-627 for a sandwich ELISA system. Thus, a hybridoma cellline can be obtained which produces the monoclonal antibody to providethe optimal combination with the anti-human-annexin-V monoclonalantibody HCA-627 for the sandwich ELISA system.

(7) Studies on Reactivities with the Solid-Phases, Sensitized withAnnexin-V Antigens Derived from Various Animals

Studies were made on animal-dependent reactivities of the variousanti-annexin-V monoclonal antibodies identified by the aforesaidscreening, by allowing them to react with microtiterplate solid phasescomposed of annexin-V antigens extracted and purified from variousanimals.

(8) Preparation of Microtiterplates with Solid Phase-Coupled NativeAnnexin-V Antigens

Native annexin-V antigens extracted and purified from the human heart,the dog heart, the rat heart and the bovine lung were each adjusted with0.1M phosphate buffer at pH 7.5 to give an antigen solution of 1microgram/ml. Each antigen solution was distributed in a 96-wellmicrotiterplate (available from Nunk Co.) at a rate of 50 microlitersper well, followed by adsorption at 4° C. overnight, washing three timeswith the washing solution and then blocking at 4° C. overnight with theblocking solution. Thus, the solid phase wells sensitized with variousannexin-V antigens were prepared for use.

Studies were made of the anti-annexin-V monoclonal antibodies producedby the hybridoma cell lines obtained by the above described screening,with respect to their reactivities with the various annexin-V antigensbound to the plates as solid phase. Thus, reaction was performed, atroom temperature for one hour, of the respective culture supernatants ofhybridoma cell line clones producing the various anti-annexin-Vmonoclonal antibodies (the original solutions) or their purified IgGfractions (prepared to give a final concentration of 1 microgram/ml)with the respective solid phase wells sensitized with the nativeannexin-V antigens derived from the various animals, followed by washingthree times with the washing solution. A further reaction was carriedout with HRPO-labeled anti-mouse-immunoglobulin antibodies (derived fromgoats) at room temperature for thirty minutes, followed by washing fourtimes with washing solution and reaction with OPD substrate solution(containing 0.1M phosphate-citrate 4 mM H₂ O₂) at room temperature forthirty minutes. Then the reaction was terminated with 2N H₂ SO₄ solutionfor absorbance assay at the primary wavelength 492 nm and the secondarywavelength 690 nm on an ELISA plate reader. Measurements at the primarywavelength 492 nm are attributed to the substrate chromogen produced bythe bound labeled-antibody, whereas the measurements at the secondarywavelength reflects the detection of flaws or stains on themicrotiterplate.

By subtracting an absorbance measurement at the secondary wavelengthfrom an absorbance measurement at the primary wavelength, it is possibleto determine the true absorbance in response to the amount of chromogenchanged due to the enzyme on the labeled-antibodies which have beenbound to antigen in proportion to the amount of the antigen bound to thesolid-phase.

(9) Cloning

Based on the absorption test using the native human- and dog-annexin-Vand through the above described capture ELISA system, the selection wasmade of hybridoma cell line clones which produce anti-annexin-Vmonoclonal antibodies to provide a prospective sensitive sandwich ELISAsystem for assaying native annexin-V in combination with theanti-annexin-V monoclonal antibody produced by hybridoma cell lineHCA-627 strain (deposited under the number FERM PB-5284 at the NationalInstitute of Bioscience and Human-Technology,the Agency of IndustrialScience and Technology, Japan). The hybridoma cell line clones weresubjected to a cloning operation by means of limiting dilution to obtaina single clone. In performing the cloning, thymus cells prepared fromBALB/c mice and suspended in HAT medium was added as feeder cells at1×10⁶ per well.

(10) Identification of Mouse Immunoglobulin Subclass

Identification of mouse immunoglobulin subclass was made with respect tothe monoclonal antibodies produced by the hybridoma cell lines whichhave been prepared as having a single clone by means of theabove-mentioned cloning. The culture supernatants of the respectivehybridoma cell lines were assayed for the mouse immunoglobulin subclassidentification using MONOAb typing kit (available from Zymed Co.).

(11) Establishment of Hybridoma Cell Lines Producing Anti-Annexin-VMonoclonal Antibodies

As a result of the first screening for the subject cell fusion, 200strains were obtained which were positive in the absorption test usingthe native human- and dog-annexin-V antigen. In addition, the secondscreening through the above described capture ELISA were selected 8clones, which can establish the sandwich ELISA system in combinationwith anti-annexin-V monoclonal antibody produced by hybridoma cell lineHCA-627 strain deposited as an International Deposit under the numberFERM BP-5284. Cloning through the limiting solution was conducted withregard to said eight elones to establish the respective hybridoma cellclones. Table 4 below gives reaction specificities of the anti-annexin-Vmonoclonal antibodies obtained by the subject cell fusion, No.2.

In table 4 there are given, for comparison, the reactivities of clonesHCA-627, HCA-155H, HCA-507HD and HCA-293HDR obtained in the previouscell fusion of Example 1.

Different from the clones established in Example 1 (HCA-115H, HCA-507HDand HCA-293HRD), all the anti-annexin-V monoclonal antibodies producedby hybridoma cell line clones established in the subject Exampledemonstrate to have a high reactivity with the native human- anddog-annexin-V (cf. the absorption tests in Table 4).

It is also shown from the screening by the capture ELISA system that theclones are able to provide an efficient and sensitive sandwich ELISAsystem in combination with the anti-annexin-V monoclonal antibodyproduced by hybridoma cell line clone HCA-627 strain (deposited as anInternational Deposit under the number FERM BP-5284) (cf. the CaptureMethod in Table 4).

In addition, from the studies of the reaction system in which annexin-Vantigens from humans and the various animals are bound as solid phase itwas found that the hybridoma cell line clones established in the subjectExample have a reactivity as monoclonal antibody totally different fromsome of the clones established in Example 1, HCA-115H, HCA-507HD andHCA-293HDR, as they do not exhibit any reactivity with the antigens ofbound annexin-V as the solid phase derived from such animals (cf. Table4), although they exhibit a strong reactivity with native annexin-V inthe absorption test, i.e. with the antigens in liquid phase.

As can be seen from the above, the eight strains as screened by thecapture ELISA system, anti-annexin-V monoclonal antibody-producingclones, are all capable of providing a sandwich ELISA system incombination with the monoclonal antibody clone HCA-627 (Table 4) andrecognizing an epitope totally different from one to be recognized bythe clone HCA-627.

As given in Table 4, the immunoglobulin subtypes of the anti-annexin-Vmonoclonal antibodies produced by the eight strains of hybridoma cellline clones established in the subject Example are all IgG type, withrespect to the H chain. Regarding the L chain all the clones havek-chain (cf. Table4).

                                      TABLE 4                                     __________________________________________________________________________    Reaction Specificities of Anti-annexin-V Monoclonal Antinodies                __________________________________________________________________________                                           Mab Screening by                                                              Mab Capture ELISA                                     Absorption Test                                                                           Absorption Test                                                                           using Anti-mouse-                                     with Native Human                                                                         with Native Dog                                                                           IgG-Fc Solid-phase                     Cell fusion    Annexin-V Antigen                                                                         Annexin-V Antigen                                                                         in Combination                         No. 2          Mab Reaction                                                                              Mab Reaction                                                                              with HRPO-labeled                      Anti-human-    Concentration                                                                             Concentration                                                                             HCA-627 Antibody                       and Anti-dog-                                                                        Mouse   10 μg/ml                                                                         1 μg/ml                                                                          10 μg/ml                                                                         1 μg/ml                                                                          Human-annexin-V                        annexin-V                                                                            Immunoglobluin                                                                        Absorption                                                                          Absorption                                                                          Absorption                                                                          Absorption                                                                          Concentration                          Mab clone No.                                                                        Isotype Rate (%)                                                                            Rate (%)                                                                            Rate (%)                                                                            Rate (%)                                                                            0 ng/ml                                                                           100 ng/ml                          __________________________________________________________________________    HCA-627                                                                              IgG1 k  98    81    98    81    0.011                                                                             0.032                              HCA-155H                                                                             IgG2a                                                                              k   4     1     4     2    --  --                                 HCA-507HD                                                                            IgG2a                                                                              k   7     3     7     5    --  --                                 HCA-293HDR                                                                           IgG2a                                                                              k   2     7     4     2    --  --                                 HDA-202                                                                              IgG1 k  98    80    97    78    0.011                                                                             2.830                              HDA-676                                                                              IgG1 k  100   98    100   99    0.013                                                                             6.270                              HDA-907                                                                              IgG1 k  98    75    99    79    0.010                                                                             3.220                              HDA-1018                                                                             IgG1 k  98    82    99    91    0.011                                                                             4.430                              HDA-1606                                                                             IgG1 k  97    68    98    77    0.005                                                                             2.615                              HDA-1884                                                                             IgG1 k  96    64    98    68    0.014                                                                             2.643                              HDA-1937                                                                             IgG1 k  99    98    100   99    0.012                                                                             9.080                              HDA-2003                                                                             IgG1 k  99    98    100   99    0.013                                                                             7.170                              __________________________________________________________________________    Reactivities with Solid-phase plates Sensitized with Annexin-V Antigens       from Various Animals                                                          Reactivity with                                                                         Reactivity with                                                                         Reactivity with                                                                         Reactivity with                                 Solid-phase-plate                                                                       Solid-phase-plate                                                                       Solid-phase-plate                                                                       Solid-phase-plate                               Sensitized with                                                                         Sensitized with                                                                         Sensitized with                                                                         Sensitized with                                 Native Annexin-V                                                                        Native Annexin-V                                                                        Native Annexin-V                                                                        Native Annexin-V                                Antigen from                                                                            Antigen from Dog                                                                        Antigen from Rat                                                                        Antigen from                                    Human Heart 50 μl/                                                                   Heart 50 μl/well                                                                     Heart 50 μl/well                                                                     Bovine Lung 50 μl/                           well Sensitization                                                                      Sensitization                                                                           Sensitization                                                                           well Sensitization                              with 1 μg/ml Antigen                                                                 with 1 μg/ml Antigen                                                                 with 1 μg/ml Antigen                                                                 with 1 μg/ml Antigen                         Reaction of 1 μg/ml                                                                  Reaction of 1 μg/ml                                                                  Reaction of 1 μg/ml                                                                  Reaction of 1 μg/ml                          Mab       Mab       Mab       Mab                                             A492 nm-A690 nm                                                                         A492 nm-A690 nm                                                                         A492 nm-A690 nm                                                                         A492 nm-A690 nm                                                                         Remarks                               __________________________________________________________________________    0.020     0.001     0.002     0.013     Established in                                                                Cell fusion No. 1                     1.986     0.001     0.006     3.540     See Table 1                                                                   Cell fusion No. 1                     2.122     0.001     0.008     3.340     See Table 1 on                                                                Cell fusion No. 1                     1.950     1.688     0.735     3.680     See Table 1 on                                                                Cell fusion No. 1                     0.007     0.000     0.000     0.000                                           0.008     0.000     0.000     0.010                                           0.012     0.005     0.000     0.001                                           0.006     0.000     0.001     0.005                                           0.010     0.000     0.001     0.002                                           0.008     0.003     0.083     0.001                                           0.007     0.004     0.003     0.014                                           0.009     0.000     0.000     0.010                                           __________________________________________________________________________     Mab: Monoclonal Antibody                                                 

EXAMPLE 9 Production of Monoclonal Antibodies

(1) Harvesting of Ascitic Fluids

In order to obtain anti-annexin-V monoclonal antibodies of a higherconcentration the various hybridoma cell lines are grown and theresulting respective clones were innoculated, following washing threetimes with EMEM culture solution, into the ascitic cavities of BALB/cmice which have been administered with 2, 6, 10, 14-tetramethylpentadecane (pristane) in advance into their ascitic cavities. Theascitic fluids were harvested seven to fourteen days after theinnoculation of the hybridoma cell lines into the ascitic casities. Theascitic fluids thus obtained were applied to a centrifuge to remove thecellular and residual fractions. The supernatant fractions were pooledfor keeping at 4° C. with the addition of sodium azide as antiseptic.

(2) Purification of Monoclonal Antibodies from Ascitic Fluids

The IgG fractions of the monoclonal antibodies were obtained by applyingthe ascitic fluids obtained from the hybridoma cell lines toaffinitychromatograpy using ConSep LC100 device and a protein-A column(available from Perceptive Co.). The purified IgG fractions of therespective clones were determined for purity by HPLC using a TSK gelG3000SW column (available from Tosoh Co.) and SDS-PAGE usingPharstSystem (available from Pharmacia Co.). The result was that all themonoclonal antibodies from the hybridoma cell lines have a purity of 98%or more.

EXAMPLE 10 Studies on ELISA System for Assay of Human Annexin-V

(1) Preparation of HRPO-Labeled Anti-Annexin-V Monoclonal Antibodies

Studies were made on the applicability of the monoclonal antibodies asestatleshed in the above to an ELISA system for assay of native humanannexin-V.

Thus, as representatives there were selected anti-annexin-V monoclonalantibodies HDA-676, HDA-907, HDA-1606 and HDA-1937, which had beenproduced by the anti-annexin-V monoclonal antibody-producing hybridomacell line clones. The selected monoclonal antibodies were applied toaffinity purification using a ConSepLC 100 device and a protein-A column(available from Perceptive Co.) to purify the IgG fractions.

(2) Preparation of Horseradish Peroxidase (HRPO) Labeled MonoclonalAntibodies

(A) Preparation of Anti-Human-Annexin-V Monoclonal Antibodies F(ab')₂

For labeling the IgG fractions of the anti-annexin-X monoclonalantibodies, the purified IgG fractions of the respective anti-annexin-Vmonoclonal antibodies HDA-676, HDA-907 (deposited as an Internationalunder the number FERM BP-5286 at the National Institute of Bioscienceand Human-Technology, the Agency of Industrial Science and Technology ofJapan), HDA-1606 and HDA-1937 were each concentrated by a Centricon 10centifugal concentrator (available from Amicon Co.) to give aconcentration of 1 ml from 10 mg. The resultant was then dialyzedagainst 0.1M sodium acetate buffer (pH 3.7) containing 0.2M NaCl assolvent. Each dialyzed IgG solution was added with pepsin in an amountof 4% of IgG for reaction at 37° C. over 2 to 4 hours. On completing thereaction, the resultant was applied to molecular-sieve chromatographyusing a Sephadex G-150 column (available from Pharmacia Co.)equilibrated with 0.1M borate buffer (pH 8.0) to obtain the F(ab')₂fragment. With every clone, 10 mg of the IgG fraction provided approx. 5mg of F(ab')₂ fraction with a high yield. There was almost no differencein yield among the clones.

(B) Preparation of Anti-Annexin-V Monoclonal Antibodies Fab'-SH

Each of the anti-annexin-V monoclonal antibodies F(ab')₂ from HDA-676,HDA-907, HDA-1606 and HDA-1937, as prepared in the above method (A), wassubjected centrifugal concentration by a Centricon 10 centrifugalconcentrator to give 1 ml of each anti-annexin-V monoclonal antibodyF(ab')₂ fraction.

To each concentrated fraction 1 ml was added with 0.1 ml of 100 mM2-mercaptoethylamine hydrochloride solution (available from KishidaChemicals Co.) for reaction at 370° C. for 90 minutes. On completing thereaction, the resultant was purified using a Sephadex G-25 column forequilibrium gel filtration measuring 1.6 cm in diameter and 20 cm inlength (available from Pharmacia Co.) and equilibrated with 0.1M sodiumphosphate buffer (pH 6.0) containing 1 mM EDTA, to fractionate theFab'-SH fraction. Following the fractionation, the resultant purifiedfraction was pooled for centifugal concentration with a Centricon 10centifugal concentrator to give a volume of 1 ml. Thus, the concentratedanti-annexin-V monoclonal antibodies Fab'-SH fractions were obtainedfrom the respective clones: HDA-676, HDA-907, HDA-1606 and HDA-1937.

(C) Preparation of HRPO Maleimide

20 mg (as protein content) of HRPO (available from Boehlinger Co.) wasdissolved in 1 ml of 0.1M sodium phosphate buffer (pH 6.0). Theresulting solution was added with 100 microliters of solution ofN-hydroxysuccinimi ester (available from Zeeben Chemicals Co.) dissolvedin dimethyl formanide (DMF ) (available from Kishida Chemicals Co.) togive a final concentration of 25 mg/l, followed by reaction at 30° C.for 60 minutes to prepare maleimide ester of HRPO. On completing thereaction, the solution was centrifuged for five minutes at 3000 rpm. Thesupernatant was applied to a Sephadex G-25 column for equilibrated gelfiltration (available from Pharmacia Co.) measuring 1.6 cm in diameterand 20 cm in length, which has been equilibrated with 0.1M sodiumphosphate buffer (pH 6.0), in order to purify HRPO maleimide. Thepurified fraction of HRPO maleimide was then subjected to centrifugalconcentration by a Centricon 10 centrifugal concentrator to prepare theconcentrated fraction of HRPO maleimide.

(D) Preparation of HRPO-Labeled Anti-Annexin-V Monoclonal AntibodiesFab'

The concentrated Fab'-SH fraction of the anti-annexin-V monoclonalantibody was mixed with the concentrated fraction of HRPO maleimide at amolar ratio of 1:1, for reaction at 4° C. for 15 to 24 hours. Oncompleting the reaction, 2-mercaptoethylamine was added to give a finalconcentration of 2 mM in the reaction solution, followed by reaction at37° C. for 20 minutes in order to block unaltered HRPO maleimide. Theresultant was then subjected to molecular-sieve gelchromatography usinga Ultragel ACA44 column (available from Pharmacia Co.) equilibrated with20 mM sodium phosphate-sodium citrate buffer (pH 5.6) containing 0.15MNaCl and 2.5M EDTA and measuring 1.6 cm in diameter and 65 cm in length,for removing unaltered Fab'-SH of the auti-annexin-V monoclonalautibodies and unaltered HRPO maleimide and purifying the respective,HRPO-labeled anti-annexin-V monoclonal antibodies Fab' (hereinafterreferred to as HRPO-labeled anti-annexin-V monoclonal antibodies).

(E) Activity Determination of HRPO-Labeled Antibodies

In determining HRPO enzyme activity of the HRPO-labeled antibodies, 2.98ml of 0.1M sodium phosphate buffer (pH 7.0) containing 0.2% phenol, 0.5mM hydrogen peroxide and 0.15 mg/ml 4-amino antipyrine was added with20-microliters of the HRPO-labeled antibody to give a total volume of3.0 ml, followed by reaction at 37° C. for five minutes, and measurementof absorbance at 500 nm by means of the Rate assay. HRPO activity wasdetermined by measuring the difference in absorbance (ΔAbs) for oneminute.

(6) Preparation of Anti-Annexin-V Monoclonal Antibody Solid PhaseCoupled Wells

In order to obtain monoclonal antibody for use as solid phase in anELISA system for assay of human annexin-V, monoclonal antibody HDA-627,produced by hybridoma cell line HCA-627 strain which has been depositedunder the number FERM BP-5284 at the International Depositary Authorityfor the deposit of microorganism, was applied to affinity purificationusing ConSepLC-100 device and a protein A column (available fromPerceptive Co.) to purify the IgG fraction from the ascitic fluid.

The IgG fraction of anti-annexin-V monoclonal antibody HCA627 wasadjusted to have a concentration of 30 micrograms/ml with 0.1M sodiumphosphate buffer (pH 7.5) containing 0.1% sodium azide, and distributedin a microtitreplate for ELISA (available from Nunk Co.) at a rate of100 microliters per well, for sensitization at 4° C. overnight. Theneach well of the microtitreplate was washed three times withphosphate-buffered saline (PBS) containing 0.05% Tween 20 surfactant,and then added with 300 microliters of PBS containing 1% BSA (as theblocking solution), followed by a further blocking operation at 4° C.overnight, to prepare antibody-plates sensitired withanti-human-annexin-V monoclonal autibodies (hereinafter referred to asauti-human-annexin-V monoclonal antibody plates).

(7) Studies on ELISA Assay System Using Anti-Annexin-V MonoclonalAntibodies

The anti-annexin-V monoclonal antibody plates as prepared above wereadded, following discarding the blocking solution, with phosphate bufferof pH 7,0 (reaction buffer) containing 1% BSA and 5 mM EDTA at a rate of100 microliters per well. Standard antigen solutions were prepared so asto give native annexin-V standard antigen concentrations of 1.563 ng/ml,3.125 ng/ml 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml and 100 ng/ml.The respective standard antigen solutions were added to the wells at arate of 20 microliters per well, followed by stirring and reaction atroom temperature for one hour. On completing the reaction, each well waswashed three times with the washing solution. Then, to the wells wereadded the HRPO-labeled anti-annexin-V monoclonal antibodies Fab'(clones: HDA-676, HDA-907, HDA-1606 and HDA-1937) at a rate of 100microliters per well, for reaction at room temperature for thirtyminutes. Following the reaction, each well was washed six times with thewashing solution and then added with 100 microliters of OPD substratesolution containing 0.1M phosphate-citrate buffer added with 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂, for reaction for thirty minutes. Thereaction was terminated by adding 2N H₂ SO₄ solution at a rate of 100microliters per well. Absorbance was determined on an ELISA plate readerat the primary wavelength 492 nm and at the secondary wavelength 690 nm.

The absorbance herein intended is obtained by subtracting an absorbancemeasurement at the secondary wanelength 690 nm from that at the primarywanelength 492 nm.

The results of absorbance measurements with respect to therepresentative native annexin-V standard antigen solutions were given inTable

                  TABLE 5                                                         ______________________________________                                        Combination of Monoclonal Antibodies in Assay of Native Human                 Annexin-V Antigen by Monoclonal Antibodies ELISA system:                      Combination of HRPO-labeled HDA-907 Antibody and HCA-627                      Solid-phase (Absorbance = A492 nm-690 nm)                                     Concentration                                                                 of Native                        Stan-                                        Human                            dard  Stan-                                  Annexin-V                        Devia-                                                                              dard                                   Antigen  Number of Measurements  tion  Error                                  (ng/ml)  1      2      3    4    Average                                                                             (SD)  CV (%)                           ______________________________________                                         0       0.006  0.007  0.007                                                                              0.006                                                                              0.007 0.0006                                                                              8.88                             1.5625   0.080  0.080  0.077                                                                              0.077                                                                              0.079 0.0017                                                                              2.21                             3.125    0.164  0.165  0.167                                                                              0.167                                                                              0.166 0.0015                                                                              0.90                             6.25     0.321  0.320  0.314                                                                              0.324                                                                              0.320 0.0042                                                                              1.31                             12.5     0.614  0.632  0.615                                                                              0.631                                                                              0.623 0.0098                                                                              1.58                             25       1.126  1.152  1.113                                                                              1.124                                                                              1.129 0.0165                                                                              1.46                             50       2.047  2.061  2.096                                                                              2.106                                                                              2.078 0.0285                                                                              1.35                             100      4.120  3.940  4.180                                                                              3.870                                                                              4.028 0.1464                                                                              3.63                             ______________________________________                                    

FIG. 12 illustrates a calibration curve for human annexin-V obtained byplotting the measurements on the human annexin-V antigen solutions withstandard antigen solution absorbance as ordinate and human annexin-Vantigen concentration as abscissa.

The calibration curve as shown in FIG. 12 is excellent one, as itdemonstrates that the absorbance rises with increasing concentration ofhuman annexin-V, substantially linearly from a low concentration up toas high as 100 ng/ml.

The calibration curve also indicates that the subject example enjoys awider dynamic range and hence provides a more sensitive system, ascompared with the calibration curve for native human annexin-V antigenin which there was employed the combination of the monocloval antibodyas solid phase produced by hybridoma cell line clone HCA-627 straindeposited under the number FERM BP-5284 as an International Deposit withHRPO-labeled anti-32KP polyclonal Fab' antibody.

It should be noted, however, that the combination of the anti-annexin-Vmonocloval antibody HCA-627 as solid phase coupled to wells and theHRPO-labeled anti-dog-32KP annexin-V polyclonal Fab' antibody is notproblematic at all in sensitivity for assay of human annexin-V.

The use of the calibration curve as obtained in the subject example(FIG. 12) enabled us to assay annexin-V concentration in the blood,plasma and serum in a highly reproducible and reliable manner from a lowconcentration to a high concentration, in which there was employed acombination of the monoclonal antibody produced by hybridoma cell lineHCA-627 strain deposited as an International Deposit under the numberFERM BP-5284 with the HRPO-labeled monocloval antibody produced byhybridoma cell line HDA-907 strain also deposited as an InternationalDeposit under the number FERM BP-5286.

The calibration curve of FIG. 12 was obtained by employing an extremelylow concentration of HRPO-labeled anti-annexin-V monoclonal antibodyHDA-907-Fab' which may have a HRPO activity of 3 to 4 mU/ml. It is thuspossible to provide a more sensitive system.

As can be seen from Table 5 and FIG. 12 on the calibration curveobtained with the standard antigen, the CV values are within 3.6% forfour times of measurements of the standard antigen concentration in therange of from a human annexin-V concentration as low as l ng/ml up to ashigh as 100 ng/ml, demonstrating the system enables a highlyreproducible assay of annexin-V concentration. From the foregoing, thesubject ELISA system using the aforesaid monoclonal antibodies for assayof annexin-V will be fully satisfactory in use as an ELISA system todetermine native annexin-V concentration in the human blood.

As shown in FIGS. 1 through 8, in a case where native human annexin-Vantigen without 8M-urea-treatment was used as standard antigen in anELISA system using the combination of the monoclonal antibodies, theabsorbance was very small, for example, as in Example 1, only about 5%to about 10% of 8M-urea-treated antigen. By contrast, the ELISA systemusing the monoclonal antibodies as obtained in the subject example hasmade a remarkable improvement in reactivity with native human annexin-Vantigen.

It was found that the HRPO-labeled Fab' antibodies of the anti-annexin-Vmonoclonal antibody clones (HDA-676, HDA-907, HDA-1606 and HDA-1937)having a reactivity with human- and dog-annexin-V and selected by thescreening operation in the subject example all exhibit a high reactivitywith human annexin-V and therefore provide a good combination withHCA-627 as shown in Example 1 in obtaining an excellent calibrationcurve for human annexin-V antigen concentration.

(8) Possibility of Detection and Quantitative Analysis of HumanAnnexin-V in Samples by Sandwich ELISA in Which Anti-Human-Annexin-VMonocloval Antibody (HCA-627) and Anti-Native-Human- and Dog-Annexin-VMonocloval Antibody Clone (HDA-907) Are Used

From among possible combinations of the monoclonal antibodies asprepared above for use in sensitive assay of native human annexin-V, asa representative was selected the combination of anti-annexin-Vmonocloval antibody HCA-627 as solid phase coupled to wells andHRPO-labeled anti-annexin-V monocloval antibody HDA-907-Fab to study itsapplication to assay of clinical samples by ELISA.

Thus, into the anti-human-annexin-V monoclonal antibody HCA-627-coupledplates was added, after discarding the blocking solution, reactionbuffer solution (sodium phosphate buffer at pH 7.0 containing 1% BSA and5 mM EDTA)at a rate of 100 microliters per well. An antigen standardsolution of native human cardiac mustle-derived annexin-V (0 ng/ml,1.5625 ng/ml, 3.125 ng/ml, 6.25 ng/ml, 12.5 ng/ml, 25 ng/ml, 50 ng/ml or100 ng/ml) and a human plasma sample are added each at a rate of 20microtiters per well, followed by stirring and reaction for one hour atroom temperature. Each well was washed four times with the washingsolution. Then there was added the HRPO-labeled anti-annexin-Vmonoclonal antibody HDA-907-Fab' having been prepared to have aconcentration of 4 mU/ml as HRPO enzyme activity, at a rate of 100microliters per well for reaction for thirty minutes at roomtemperature, followed by washing four times with the washing solution.After the washing the wells were added with OPD substrate solution(containing 0.1M phosphate-citrate buffer added with 2 mg/mlo-phenylenediamine and 4 mM H₂ O₂) at a rate of 100 microliters perwell, followed by absorbance measurements at the primary wanelength 492nm and at the secondary wanelength 690 nm on an ELISA plate reader.

The human plasma samples were determined for native human annexin-Vconcentration by reading the calibration curve which was prepared byplotting the absorbance against varying concentration of human cardiacmuscle-derived annexin-V as standard antigen. The data for thecalibration curve are given in Table 5 and FIG. 12. As mentionedpreviously, the absorbance increased with increasing concentration ofthe human annexin-V standard antigen.

(9) Sample Dilution Tests

Dilution tests were performed on a plasma sample prepared by addingpurified annexin-V antigen to normal plasma(Sample 1), a plasma samplewith a low value of annexin-V concentration (Sample 2) and a plasmasample with a high value of annexin-V concentration (Sample 3). Eachsample was subjected to a series of successive doubling dilution. Eachseries of samples, including the original sample solution, were assayedfor annexin-V concentration by the ELISA system for annexin-V assayusing the combination of the monoclonal antibody clone HCA-627 as solidphase coupled to well and the HRPO-labeled monoclonal antibody cloneHDA-907-Fab'.

The results are given in Table 6, Tables 6-1 through 6-3 and FIG. 13.

                  TABLE 6                                                         ______________________________________                                        Results of Sample Dilution Tests (Measurements n = 2)                         Sample Dilution                                                                            Human Annexin-V Antigen Measurements                             Dilution                                                                             1/Dilution                                                                              (ng/ml)                                                      Rate   Rate      Sample 1   Sample 2                                                                              Sample 3                                  ______________________________________                                         × 32                                                                          0.03125   0.97               1.13                                       × 16                                                                          0.0625    2.37       0.20    2.71                                      × 8                                                                            0.125     4.86       0.56    5.87                                      × 4                                                                            0.25      9.22       1.59    11.98                                     × 2                                                                            0.5       17.85      3.52    22.63                                     × 1                                                                            1         35.80      6.95    42.94                                     ______________________________________                                    

                  TABLE 6-1                                                       ______________________________________                                        Data on Sample 1                                                              (A plasma sample prepared by adding human annexin-V                           to normal plasma)                                                             Sample Dilution                                                                          Dilution                                                                              Number of Measurements                                                                         Average                                   Rate       Factor  1          2       (ng/ml)                                 ______________________________________                                         × 32                                                                              0.03125 0.95       0.99    0.97                                     × 16                                                                              0.0625  2.37       2.36    2.37                                    × 8  0.125   4.89       4.82    4.86                                    × 4  0.25    9.33       9.10    9.22                                    × 2  0.5     18.50      17.20   17.85                                   × 1  1       37.00      34.60   35.80                                   ______________________________________                                    

                  TABLE 6-2                                                       ______________________________________                                        Data on Sample 2                                                              (A sample having a high value of human annexin-V antigen                      concentration: KAN-1)                                                         Sample Dilution                                                                          Dilution                                                                              Number of Measurements                                                                         Average                                   Rate       Factor  1          2       (ng/ml)                                 ______________________________________                                         × 32                                                                              0.03125                                                             × 16                                                                              0.0625  0.19       0.21    0.20                                    × 8  0.125   0.50       0.61    0.56                                    × 4  0.25    1.54       1.63    1.59                                    × 2  0.5     3.51       3.53    3.52                                    × 1  1       6.94       6.96    6.95                                    ______________________________________                                    

                  TABLE 6-3                                                       ______________________________________                                        Data on Sample 3                                                              (A sample having a high value of human annexin-V antigen                      concentration: KAN-2)                                                         Sample Dilution                                                                          Dilution                                                                              Number of Measurements                                                                         Average                                   Rate       Factor  1          2       (ng/ml)                                 ______________________________________                                         × 32                                                                              0.03125 1.10       1.16    1.13                                     × 16                                                                              0.0625  2.63       2.78    2.71                                    × 8  0.125   5.77       5.97    5.87                                    × 4  0.25    11.94      12.01   11.98                                   × 2  0.5     22.46      22.79   22.63                                   × 1  1       42.16      43.72   42.94                                   ______________________________________                                    

With Sample 1, a sample prepared by adding annexin-V to normal plasma,the annexin-V concentration in blood varies with the dilution rate,forming a straight dilution line directing toward the origin. With bothSample 2 and Sample 3 (a sample having a high annexin-V concentration),the annexin-V measurement varies in a close relationship with thedilution rate, also forming a substantially straight dilution linedirecting toward the origin.

Thus, as it was found that the annexin-V measurement is closely relatedwith the dilution rate, forming a straight dilution line directingtoward the origin for all samples with or without annexin-V addition,the subject system can provide an assay system for quantitating theannexin-V concentration in samples without interference by possiblecomponents in the plasma.

In addition, for the purpose of confirming the reliability of thesubject system for assay of clinical samples, recovery tests wereconducted by adding human annexin-V as purified annexin-V antigen toplasma and serum samples.

(10) Human Annexin-V Recovery Tests in ELISA System for Assay of HumanAnnexin-V

With respect to the ELISA system as studied above in which there areused anti-annexin-V monoclonal antibody HCA-627 as solid phase coupledto wells and HRPO-labeled anti-annexin-V monoclonal antibodyHDA-907-Fab', recovery tests for human annexin-V were conducted toexamine whether purified annexin-V antigens as added to plasma or serumare recovered without any influence on the measurements of annexin-V bycomponents present in the plasma or serum.

(11) Procedure for Recovery Test

Purified human annexin-V antigen solutions were prepared to have aconcentration of 40.8 ng/ml, 14.6 ng/ml and 4.7 ng/ml. To each of threehuman plasma samples and two human serum samples was added one of thepurified human annexin-V antigens having the three different(high,middle and low) concentrations in a proportion of 9 volumes of sample to1 volume of antigen to observe annexin-V concentration in each sample.The human annexin-V antigen solution was replaced by buffer solutionwhich was also added to each sample in the same proportion as in thecase of human annexin-V antigen, so as to observe human annexin-Vconcentration in each sample per se. Human annexin-V antigenconcentration as added was determined through assay of a sample-likesolution prepared by adding human annexin-V antigen to reaction buffersolution instead of the plasma or serum sample in the same proportion asin such sample.

The concentration of human annexin-V recovered was obtained bysubtracting measured human annexin-V concentration of a sample per sefrom measured human annexin-V concentration as added, as given by thefollowing equation(1):

    The concentration of annexin-V antigen recovered (ng/ml)=Measured human annexin-V concentration of a sample added with the antigen of the respective concentration (ng/ml)--Measured human annexin-V concentration of the per se (ng/ml)                                     (1)

The recovery rate at the concentration of annexin-V recovered wasobtained by the following equation (2):

    Recovery Rate (%)=(Concentration of human annexin-V recovered when added with the antigen of the respective concentration/Human annexin-V concentration as added)×100                         (2)

The results of the recovery tests are summarized in Table

                                      TABLE 7                                     __________________________________________________________________________    Results of Recovery Tests                                                                                    Measured Human Annexin-V                                                      Antigen Concentration in                                                      Sample When Added with                                          Measured                                                                             Human  Human Annexin-V Antigen                                Measured Human  Annexin-V                                                                            of the Respective                              Sample  Human Annexin-V                                                                        Annexin-V                                                                            Antigen                                                                              Concentration Human                                Identifi-                                                                         Concentration                                                                          Concentration                                                                        Concentration                                                                        Measure-                                                                           Measure- Annexin-V                        Type of                                                                           cation                                                                            in Sample per                                                                          as Added                                                                             Recovered                                                                            ment 1                                                                             ment 2                                                                             Average                                                                           Recovery                         Sample                                                                            No. se (ng/ml)                                                                             (ng/ml)                                                                              (ng/ml)                                                                              (ng/ml)   (ng/ml)                                                                           Rate (%)                         __________________________________________________________________________    Plasma                                                                            KAN-1                                                                             6.27     4.70   4.96   11.16                                                                              11.30                                                                              11.23                                                                             105.5                                             14.60  14.08  20.10                                                                              20.59                                                                              20.35                                                                             96.4                                              40.81  39.64  46.18                                                                              45.63                                                                              45.91                                                                             97.1                             Plasma                                                                            KAN-2                                                                             42.00    4.70   5.11   45.30                                                                              48.91                                                                              47.11                                                                             108.6                                             14.60  15.37  55.82                                                                              58.91                                                                              57.37                                                                             105.2                                             40.81  44.84  85.28                                                                              88.40                                                                              86.84                                                                             109.9                            Plasma                                                                            TK  1.27     4.70   5.01   6.14 6.41 6.28                                                                              106.5                                             14.60  14.98  15.98                                                                              16.52                                                                              16.25                                                                             102.6                                             40.81  39.07  39.34                                                                              41.34                                                                              40.34                                                                             95.7                             Serum                                                                             93011                                                                             1.59     4.70   4.75   6.39 6.28 6.34                                                                              101.0                                             14.60  13.38  14.97                                                                              14.96                                                                              14.97                                                                             91.6                                              40.81  36.93  38.96                                                                              38.08                                                                              38.52                                                                             90.5                             Serum                                                                             93025                                                                             1.57     4.70   4.96   6.55 6.51 6.53                                                                              105.5                                             14.60  14.42  15.98                                                                              16.00                                                                              15.99                                                                             98.8                                              40.81  39.94  40.92                                                                              42.09                                                                              41.51                                                                             97.9                             __________________________________________________________________________

The result of the recovery test was excellent in the plasma samples withfrom a low value (1.27 ng/ml) up to a high value (42.0 mg/ml) ofannexin-V concentration in sample per se, as the antigen recovery ratewas between 96% and 109% in the concentration range of human annexin-Vantigen added (from 4.7 ng/ml to 40.8 ng/ml).

The recovery rate was also excellent with respect to the serum samples,as it was between 91% and 106% in the concentration range of humanannexin-V antigen added, i.e. from a low concentration (4.7 ng/ml) up toa high concentration (40.8 ng/ml).

As can be seen from the foregoing, the subject ELISA system for assay ofannexin-V has a good prospect of application to the assay of clinicalsamples because it has enabled reliable quantitative analysis withoutbeing influenced by components of the plasma and serum.

EXAMPLE 11 Application of the Present Invention to Analysis of BloodSample by Enzyme Antibody Method

(1) Pretreatment of Collected Blood

Intvavenuously collected whole blood was placed without delay into a 1.8ml collecting tube containing 0.2 ml of 3.8% sodium citrate, followed bymild stirring and centrifugation at 2500 rpm for 10 minute to isolateplasma. The isolated plasma was immediately or, following after frozenfor keeping until use for keeping, supplied for use in the assay ofannexin-V concentration.

In the case when EDTA salt is used in place of sodium citrate, 3 to 5 mMEDTA 2 potassium or EDTA 2 sodium may be placed in the blood collectingtube to isolate plasma following centrifugation.

(2) Diagnosis of Myocardial Infarction

Analysis of blood collected three hours after the onset from a72-year-old lady and having no symptom of hemolysis indicated CK(creatine phosphate enzyme) value of 50 U/L, a normal value, andannexin-V concentration in plasma of 16.4 ng/ml, an increased value. Thecase was later affirmatively diagnostic of myocardial infection becauseof elevation of CK value up to 1108 U/L and a typical abnormality on theelectrocardiogram. Annexin-V concentration in plasma returned to anormal value three days after the onset of the disease.

This case indicates that the assay of annexin-V concentration in plasmain the first sampling of blood with no hemolysis is of use in diagnosisof myocardial infarction. Annexin-V concentration in plasma of a patientwith myocardial infection is relatively high in an early stage of thedisease (within 6 hours after the onset of attack). From the analysis ofblood samples without homolysis from patients complaining of pains inthe chest, it was later established that 70% of the cases, whereannexin-V concentration in blood was determined to show 10 ng/ml ormore, is diagnostic of myocardial infarction.

(3) Diagnosis of Angina Pectoris

Blood with no hemolysis from a 55-year-old man who complained of a painin the chest three hours after a first pain, was collected three and ahalf hours after the onset and analyzed for annexin-V concentration inplasma, with the result that the concentration was 5.8 ng/ml. Analysiswas also conducted on blood samples from the same patient thereafter forCK values in the blood with the result that the values were 140 U/L orless, all a normal value. Coronary angiography taken later showed a highdegree of stenosis on the right coronary artery. Thus, based on thisfact with reference to the clinical symtoms, the case was affirmativelydiagnosed as angina pectoris.

This case demonstrates that the analysis of blood with no hemolysis todetermine annexin-V concentration in plasma is of use in the diagnosisof angina pectoris. While annexin-V concentration in plasma of a patientwith annexin-V angina pectoris is relatively low as compared with a caseof myocardial infarction, approx. 70% of cases, where CK value of apatient complaining of pains in the chest was not diagnostic of anginapectoris but the annexin-V concentration in plasma showed 5 ng/ml ormore, were diagnosed as angina pectoris.

(4) Annexin-V Concentration in Normal Adults

Annexin-V concentration in plasma from blood with no hemolysis of normalmales 22 to 75 years of age is in the range of 0.6 to 1.9 ng/ml, whereasthat of normal females 24 to 78 years of age is in the range of 0.5 to1.7 ng/ml, with the average being 1.0 ng/ml, the maximum 1.9 ng/ml andthe minimum 0.5 ng/ml. There was observed neither sex-dependent norage-dependent variations.

Hemolysis will bring a higher value of annexin-V concentrationmeasurement in plasma because annexin-V leaks into the plasma from thedecomposed leukocytes, platelets and erythrocytes. In treating bloodsample following the collection, care should therefore be taken not tocause hemolysis to occur, for example, through use of a fine collectingneedle or avoidance of vigorous shaking of blood samples. Slighthemolysis will give no substantial influence on the assay of annexin-Vconcentration in blood, enabling such assay. Thus, the assay ofannexin-V concentration in plasma is of use in the diagnosis ofmyocardial and angina pectoris.

(5) Comparison with Example 7

In Example 7 the numerical values of analysis, including the normalvalues, are somewhat higher than those in Example 9. This is because theanalysis in Example 7 includes measurement of annexin-V concentrationdue to hemolysis which have occurred during the treatment of the bloodsamples.

Example 7 therefore suggests that, as long as blood samples are treatedin the same manner, assay of annexin-V concentration in plasma is of usein the diagnosis of myocardial infarction and angina pectoris even ifhemolysis occurs.

(6) As can be seen from the description of the invention,anti-human-annexin-V monoclonal antibody HCA-627 andanti-human-annexin-V monoclonal antibody HDA-907 obtained by the presentinvention are monoclonal antibodies which are capable of specificallyrecognizing an annexin-V antigenic molecule through different antigenicdeterminants on an identical annexin-V molecule.

The sandwich ELISA system using the two monoclonal antibodies enablesthe assay of annexin-V antigen in a highly sensitive manner, providingexcellent quantitative analysis as can be seen from the satisfactoryresults of the dilution tests and the recovery tests.

In addition, the two monoclonal antibodies are capable of cross-reactingwith both human-annexin-V antigen and dog annexin-V antigen. The twocross-reacting monoclonal antibodies are considered to recognizeantigenic determinants common to the different types of annexin-Vantigen molecules. Thus, in preparing standard antigen for assayannexin-V, such monoclonal antibodies make it possible to provide lessexpensive dog-derived annexin-V without causing the ethical issue withrespect to extraction and purification of human-derived annexin-V.

Industrial Applicability

The present invention is directed to the production of novelanti-human-annexin-V monoclonal antibodies by culturing hybridoma celllines HCA-627 strain (deposited on Nov. 6, 1995 as an InternationalDeposit under the number FERM BP-5284 at the National Institute ofBioscience and Human-Technology, the Agency of Industrial Science andTechnology of Japan, at Higashi 1-1-3, Tukuba-City, Ibaraki-Pref.,Japan, an International Depositary Authority for the deposit ofmicroorganisms) and HDA-907 strain (deposited on Nov. 7, 1995 as anInternational Deposit under the number FERM BP-5286 at the NationalInstitute of Bioscience and Human-Technology, the Agency of IndustrialScience and Technology of Japan, at Higashi 1-1-3, Tukuba-City,Ibaraki-Pref., Japan, an International Depositary Authority for thedeposit of microorganisms), in which the hybridoma cells were preparedby extracting lymphocytic plasmablast cells from lymphoid organs ofmammalian animals such as mice immunized with human annexin-V, anantigenic protein from human heart, and fusing lymphocytic plasmablastcells with myeloma cells.

The anti-human-annexin-V monoclonal antibodies obtained in the presentinvention are ones capable of recognizing specifically human annexin-Vas the antigenic protein.

In addition to the anti-human-annexin-V monoclonal antibodies there canbe also obtained, according to the present invention, anti-annexin-Vmonoclonal antibodies which will cross-react with annexin-V as antigenicprotein derived from various animals (e.g. beagles and rats).

These cross-reacting anti-annexin-V monoclonal antibodies are consideredto have an ability to recognize antigenic determinant sites occurringcommonly on the annexin-V protein molecules. Such sites having antigenicdeterminants common to the annexin-V protein molecules are importantones for maintaining the molecular structure of annexin-V reserved amongdifferent animal species.

The use of the anti-human-annexin-V monoclonal antibodies of the presentinvention makes it possible to diagnose patients with ischemic diseasessuch as myocardial infarction and angina pectoris by means of ELISA, forexample, through the measurement of human annexin-V concentration in theblood. Thus, although conventionally thought impossible, myocardialinfarction and angina pectoris can be diagnosed quickly and reliablyeven at the early stages of the diseases, and the reliable diagnosis canbe conducted free of influence by rheumatoid factor.

Another exemplarly advantage of the present invention is that it can beapplied in autopsy or post mortem to stain the cardiac muscle tissueusing the anti-human-annexin-V monoclonal antibodies so as to detect thedistribution of human annexin-V through the cardiac muscle tissue.

In a further aspect of the present invention, there can be obtainedanti-human-annexin-V monoclonal antibodies which are different inreactivity and are capable of recognizing different antigenicdeterminants on a human annexin-V molecule. By selecting, from a groupof such anti-human-annexin-V monoclonal antibodies, two different typesof anti-human-annexin-V monoclonal antibodies different from each otherin antigenic determinant for recognition and combining them, it ispossible to determine human annexin-V with a high sensitivity andreproducibility by means of ELISA.

Thus, by the combination of the anti-human-annexin-V monoclonalantibodies having different specificities to different antigenicdeterminants, preparation is now possible of highly reliable reagentsfor the assay of myocardial infarction and angina pectoris.

The anti-human-annexin-V monoclonal antibodies of the present inventionare also suitable for use in tissue-staining, in addition to their usein the ELISA system for the assay of human annexin-V. Through thetissue-staining analysis, it is possible to identify the areas in themyocardial tissue of human heart where human anexin localizes ordistributes, and of the areas where human annexin-V passes from themyocardial tissue due to ischemia, thereby greatly contributing tomolecular physiological and other studies. It is also possible torecover annexin-V molecules of a high purity from the tissue extractsthrough the purification by means of affinity chromatography using theanti-human-annexin-V monoclonal antibodies or the anti-annexin-Vmonoclonal antibodies. Further, identification of annexin-V moleculescan be made by means of Western blotting.

As can be seen from the above, the anti-human-annexin-V monoclonalantibodies and the anti-annexin-V monoclonal antibodies obtained fromvarious animals according to the present invention can be used inmolecular and physiological studies on annexin-V occurring in humans andvarious animals, thereby making a great contribution in the wide rangeof areas including both basic medicine and clinical medicine.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 2                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GluTyrGlySerSerLeuGlu                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GlyThrAspGluGluLysPheIleThrIlePheGlyThr                                       1510                                                                          __________________________________________________________________________

We claim:
 1. An anti-annexin-V monoclonal antibody produced by ahybridoma cell line which is selected from the group consisting of thehybridoma cell lines deposited as FERM BP-5284 and FERM BP-5286 at theInternational Depository Authority for the deposit of microorganisms,said anti-annexin-V monoclonal antibody being cross-reactive withannexin-V from the heart cells of one or more mammalian species.
 2. Ahydridoma cell line selected from the group consisting of the hybridomacell lines deposited as FERM BP-5284 and FERM BP-5286 at theInternational depository Authority for the deposit of microorganisms,said hybridoma cell line having the ability to produce an anti-annexin-Vmonoclonal antibody which has a binding specificity to an antigenicdeterminant on annexin-V antigenic protein and which is capable ofcross-reacting with annexin-V from the heart of one or more mammalianspecies.
 3. A diagnostic agent for myocardial infarction and anginapectoris which comprises:a first reagent containing a firstanti-annexin-V monoclonal antibody being produced by a hybridoma cellline selected from the group consisting of the hybridoma cell linesdeposited as FERM BP-5284 and FERM BP-5286 at the InternationalDepository Authority for the deposit of microorganisms; a second reagentcontaining a second anti-annexin-V monoclonal antibody being produced bythe other hybridoma cell line selected from said group, or polyclonalantibody, wherein the antibody of the second reagent is labeled; whereinthe antibodies of both reagents are cross-reactive with annexin-V fromhuman heart cells and the annexin-V antigen is annexin-V from heartcells of one or more mammalian species.
 4. The diagnostic agent formyocardial infarction and angina pectoris of claim 3 in which the firstanti-annexin-V monoclonal antibody is anti-annexin-V monoclonal antibodyproduced by the hybridoma cell line FERM BP-5284 and the labeled secondanti-annexin-V monoclonal antibody is produced by labeling theanti-annexin-V monoclonal antibody produced by the hybridoma cell lineFERM BP-5286.
 5. The diagnostic agent for myocardial infarction andangina pectoris of claim 3 in which the first anti-annexin-V monoclonalantibody is anti-annexin-V monoclonal antibody produced by the hybridomacell line FERM B-5286 and the labeled second anti-annexin-V monoclonalantibody is produced by labeling the anti-annexin-V monoclonal antibodyproduced by the hybridoma cell line FERM BP-5284.
 6. The diagnosticagent for myocardial infarction and angina pectoris of claim 3 in whichthe first anti-annexin-V monoclonal antibody is anti-annexin-Vmonoclonal antibody produced by the hybridoma cell line FERM BP-5284 andthe labeled second anti-annexin-V polyclonal antibody isanti-dog-annexin-V antibody which cross-reacts with human annexin-V. 7.The diagnostic agent for myocardial infarction and angina pectoris ofclaim 3 in which the first anti-annexin-V monoclonal antibody isanti-annexin-V monoclonal antibody produced by the hybridoma cell lineFERM BP-5286 and the labeled second anti-annexin-V polyclonal antibodyis anti-dog-annexin-V antibody which cross-reacts with human annexin-V.8. A method for diagnosing myocardial infarction and angina pectoriscomprising the steps of:causing an antigen-antibody reaction ofannexin-V in a sample with a first anti-annexin-V monoclonal antibody toform an annexin-V antigen/anti-annexin-V monoclonal antibody complex;allowing the antigenic site of annexin-V of the formed annexin-Vantigen/anti-annexin-V monoclonal antibody complex to be bound with alabeled second anti-annexin-V monoclonal antibody or labeledanti-annexin-V polyclonal antibody so as to form a labeled form of saidannexin-V antigen/anti-annexin-V monoclonal antibody complex bound withthe polyclonal or monoclonal antibody; and quantitatively analyzing thelabeled form of said complex to perform said diagnosis.
 9. The method ofdiagnosing myocardial infarction or angina pectoris of claim 8 in whichthe first anti-annexin-V monoclonal antibody is produced by a hybridomacell line selected from the group consisting of the hybridoma cell linesdeposited as FERM BP-5284 and FERM BP-5286 at the InternationalDepository Authority for the deposit of microorganisms, and the labeledsecond anti-annexin-V monoclonal antibody is produced by labeling thesecond anti-annexin-V monoclonal antibody produced being different fromthe first anti-annexin-V monoclonal antibody produced by a hybridomacell line which is selected from the same said group, wherein said firstand second anti-annexin monoclonal antibodies have a binding specificityfor an antigenic determinant on annexin-V protein from human heartcells.
 10. The method for diagnosing myocardial infarction and anginapectoris of claim 8 in which the first anti-annexin-V monoclonalantibody is anti-annexin-V monoclonal antibody produced by the hydridomacell line FERM BP-5284.
 11. The method for diagnosing myocardialinfarction and angina pectoris of claim 8 in which the firstanti-annexin-V monoclonal antibody is anti-annexin-V monoclonal antibodyproduced by the hybridoma cell line FERM BP-5286.
 12. The method fordiagnosing myocardial infarction and angina pectoris of claim 8 in whichthe labeled second anti-annexin-V monoclonal antibody is anti-annexin-Vmonoclonal antibody produced by the hybridoma cell line FERM BP-5284.13. The method for diagnosing myocardial infarction angina pectoris ofclaim 8 in which the labeled second anti-annexin-V monoclonal antibodyis anti-annexin-V monoclonal antibody produced by the hybridoma cellline FERM BP-5286.
 14. A method for analyzing annexin-V in human cardiacmuscle or in a sample which comprises causing an antigen-antibodyreaction of human annexin-V in the human cardiac muscle or the samplewith an anti-annexin-V monoclonal antibody produced by a hybridoma cellline selected from the group consisting of the hybridoma cell linesdeposited as FERM BP-5284 and FERM BP-5286 at the InternationalDepository Authority for the deposit of microorganisms, to form anannexin-V antigen-anti-annexin-V monoclonal antibody complex andquantitatively analyzing the formed annexin V antigen-anti-annexin-Vmonoclonal antibody complex.
 15. A method for analyzing annexin-V in asample which comprises the steps of:causing an antigen-antibody reactionof human annexin-V in the sample with a first anti-annexin-V monoclonalantibody produced by a hybridoma cell line selected from the groupconsisting of the hybridoma cell lines deposited as FERM BP-5284 andFERM BP-5286 at the International Depository Authority for the depositof microorganisms to form an annexin-V antigen-anti-annexin-V monoclonalantibody complex, allowing the antigenic site of human annexin-V of theformed annexin-V antigenic-anti-annexin-V monoclonal antibody complex tobe bound with a labeled second anti-annexin-V monoclonal antibody oranti-annexin-V polyclonal antibody, so as to form a labeled from of saidannexin-V antigen-anti-annexin-V monoclonal polyclonal antibody complexbound with labeled anti-annexin-V polyclonal antibody or labeled thesecond anti-annexin-V monoclonal antibody; quantitatively analyzing thelabeled form of the complex and; said labeled second anti-annexinmonoclonal antibody is produced by labeling second anti-annexin-Vmonoclonal antibody being different from the first anti-annexin-Vmonoclonal antibody and produced by a hybridoma cell line which isselected from the group consisting of the hybridoma cell line depositedas FERM BP-5284 and FERM BP-5286 at the International DepositoryAuthority for the deposit of microorganisms.
 16. The method of claim 15in which the first anti-annexin-V monoclonal antibody is one produced bythe hybridoma cell line FERM BP-5284 and has a binding specificity foran antigenic determinant on human annexin-V antigenic protein.
 17. Themethod of claim 15 in which the first anti-annexin-V monoclonal antibodyis one produced by the hybridoma cell line FERM BP-5286 and has abinding specificity for an antigenic determinant on human annexin-Vantigenic protein.
 18. The method of claim 15 in which the labeledsecond anti-annexin-V monoclonal antibody is produced by labeling theanti-annexin-V monoclonal antibody produced by hybridoma cell line FERMBP-5284 and has a binding specificity for an antigenic determinant onhuman annexin-V antigenic protein.
 19. The method of claim 15 in whichthe labeled second anti-annexin-V monoclonal antibody is produced bylabeling the anti-annexin-V monoclonal antibody produced by hybridomacell line FERM BP-5286 at and has a binding specificity for an antigenicdeterminant on human annexin-V antigenic protein.